Phosphonate metabolizing plants

ABSTRACT

The invention relates in general to herbicide resistance in plants, and more particularly to a new class of phosphonate metabolizing genes, and methods of using these genes for improving plant tolerance to phosphonate herbicides.

[0001] REFERENCE TO PRIOR APPLICATIONS

[0002] This application claims the benefit of priority to U.S.Provisional Application Serial No. 60/108,763 filed Nov. 17, 1998.

FIELD OF THE INVENTION

[0003] The present invention relates in general to herbicide resistancein plants, and more particularly to a new class of phosphonatemetabolizing genes and methods of using these genes for improving planttolerance to phosphonate herbicides.

DESCRIPTION OF THE PRIOR ART

[0004] Phosphorous containing organic molecules can be naturallyoccurring or synthetically derived. Organic molecules containingphosphorous-carbon (C—P) bonds are also found naturally or as syntheticcompounds, and are often not rapidly degraded, if at all, by naturalenzymatic pathways. Synthetic organophosphonates and phosphinates,compounds that contain a direct carbon-phosphorous (C—P) bond in placeof the better known carbon-oxygen-phosphorous linkage of phosphateesters (Metcalf et al., Gene 129:27-32, 1993), have thus been widelyused as insecticides, antibiotics, and as herbicides (Chen et al., J.Biol. Chem. 265:4461-4471, 1990; Hilderbrand et al., The role ofphosphonates in living systems, Hilderbrand, R. L., ed, pp. 5-29, CRCPress, Inc., Boca Raton, Fla, 1983). Phosphonates are ubiquitous innature, and are found alone and in a diversity of macromolecularstructures in a variety of organisms (Jiang et al., J. Bacteriol.177:6411-6421, 1995). Degradation of phosphonate molecules proceedsthrough a number of known routes, a C—P lyase pathway, a phosphonatasepathway, and a C—N hydrolysis pathway (Wanner, Biodegradation 5:175-184,1994; Barry et al., U.S. Pat. No. 5,463,175, 1995). Bacterial isolatescapable of carrying out these steps have been characterized (Shinabargeret al., J. Bacteriol. 168:702-707, 1986; Kishore et al., J. Biol. Chem.262:12,164-12,168, 1987; Pipke et al., Appl. Environ. Microbiol.54:1293-1296,1987; Jacob et al., Appl. Environ. Microbiol. 54:2953-2958,1988; Lee et al., J. Bacteriol. 174:2501-2510, 1992; Dumora et al.,Biochim. Biophys. Acta 997:193-198, 1989; Lacoste et al., J. Gen.Microbiol. 138:1283-1287, 1992). However, with the exception ofphosphonatase and glyphosate oxidase (GOX), other enzymes capable ofcarrying out these reactions have not been characterized.

[0005] Several studies have focused on the identification of genesrequired for C—P lyase degradation of phosphonates. Wackett et al. (J.Bacteriol. 169:710-717, 1987) disclosed broad substrate specificitytoward phosphonate degradation by Agrobacterium radiobacter and specificutilization of glyphosate as a sole phosphate source. Shinabarger et al.and Kishore et al. disclosed C—P lyase degradation of the phosphonateherbicide, glyphosate, to glycine and inorganic phosphate through asarcosine intermediate by Pseudomonas species.

[0006]E. coli B strains had previously been shown to be capable ofphosphonate utilization (Chen et al.), whereas E. coli K-12 strains wereincapable of phosphonate degradation. However, K-12 strains weresubsequently shown to contain a complete, though cryptic, set of genes(psiD or phn) capable of phosphonate utilization (Makino et al.), asmutants were easily selected by growth on low phosphate media containingmethyl- or ethyl-phosphonate as sole phosphorous sources. Such K-12strains adapted for growth on methyl- or ethylphosphonate weresubsequently shown to be able to utilize other phosphonates as solephosphorous sources (Wackett et al., J. Bacteriol. 169:1753-1756, 1987).

[0007] Avila et al. (J. Am. Chem. Soc. 109:6758-6764, 1987) wereinterested in the mechanistic appraisal of biodegradative anddetoxifying processes as related to aminomethyl-phosphonates, includingelucidating the intermediates, products, and mechanisms of thedegradative dephosphorylation process. Avila et al. studied theformation of dephosphorylated biodegradation products from a variety ofaminophosphonate substrates in E. coli K-12 cultures previously adaptedto growth on ethylphosphonate. Furthermore, Avila et al. utilizedN-acetyl-AMPA (N-acetyl-amino-methyl-phosphonate) as a sole phosphatesource in some of their studies in order to show that acetylated AMPAwas not inhibitory to C—P bond cleavage. In addition, Avila et al. notedthat N-acetyl-AMPA was able to serve as a sole phosphate source duringE. coli K-12 growth, however, they did not observe N-acetyl-AMPAformation when AMPA was used as a sole phosphate source. Their resultsindicated that AMPA was not a substrate for acetylation in E. coli.

[0008] Chen et al. identified a functional psiD locus from E. coli B bycomplementation cloning into an E. coli K-12 strain deficient forphosphonate utilization, which enabled the K-12 strain to utilizephosphonate as a sole phosphate source (J. Biol. Chem. 265:4461-4471,1990). Chen et al. thus disclosed the DNA sequence of the psiDcomplementing locus, identified on a 15.5 kb BamHI fragment containing17 open reading frames designated phnA-phnQ, comprising the E. coli Bphn operon. The cryptic phn (psiD) operon from E. coli K-12 wassubsequently found to contain an 8-base pair insertion in phnE. Theresulting frameshift in phnE not only results in defective phnE geneproduct, but also apparently causes polar effects on the expression ofdownstream genes within the operon, which prevent phosphonateutilization (Makino et al., J. Bacteriol. 173:2665-2672, 1991). Theoperon has been more accurately described to contain the genes phnC-phnPby the work of Makino et al. Further research has been directed tounderstanding the nature of the function of each of the genes withinthis operon (Chen et al., J. Biol. Chem. 265:4461-4471, 1990; Makino etal., J. Bacteriol. 173:2665-2672, 1991; Wanner et al., FEMS Microbiol.Lett. 100:133-140, 1992; Metcalf et al., Gene 129:27-32, 1993; Ohtaki etal., Actinomyceteol. 8:66-68, 1994). In all of these efforts, the phnOgene has been implicated as a regulatory protein based on its similarityto other nucleotide binding proteins containing structuralhelix-turn-helix motifs. Furthermore, mutagenesis of genes in the phnoperon demonstrated that phnO was not required for phosphonateutilization, further supporting the proposed regulatory function forthis gene (Metcalf et al., J. Bacteriol. 173:587-600, 1991), at leastfor the phosphonates tested. Homologous phn sequences have beenidentified from other bacteria, including a gene substantially similarto E. coli phnO, isolated from S. griseus, using nucleotide sequencesdeduced from those in the E. coli phnO gene (Jiang et al., J. Bacteriol.177:6411-6421, (1995); McGrath et al., Eur. J. Biochem. 234:225-230,(1995); Ohtaki et al., Actinomyceteol. 8:66-68, (1994)). However, nofunction other than as a regulatory factor has been proposed for phnO. Aregulatory role for phnO in the CP lyase operon has been cited again ina recent review (Berlyn, Microbiol. Molec. Biol. Rev. 62:814-984, 1998).

[0009] Advances in molecular biology, and in particular in plantsciences in combination with recombinant DNA technology, have enabledthe construction of recombinant plants which contain nonnative genes ofagronomic importance. Furthermore, when incorporated into and expressedin a plant, such genes desirably confer some beneficial trait orcharacteristic to the recombinant plant. One such trait is herbicideresistance. A recombinant plant capable of growth in the presence of aherbicide has a tremendous advantage over herbicide-susceptible species.In addition, herbicide tolerant plants provide a more cost effectivemeans for agronomic production by reducing the need for tillage tocontrol weeds and volunteers.

[0010] Chemical herbicides have been used for decades to inhibit plantmetabolism, particularly for agronomic purposes as a means forcontrolling weeds or volunteer plants in fields of crop plants. A classof herbicides which have proven to be particularly effective for thesepurposes are known as phosphonates or phosphonic acid herbicides.Perhaps the most agronomically successful phosphonate herbicide isglyphosate (N-phosphono-methyl-glycine).

[0011] Recombinant plants have been constructed which are tolerant tothe phosphonate herbicide glyphosate. When applied to plants, glyphosateis absorbed into the plant tissues and inhibits aromatic amino acidformation, mediated by an inhibition of the activity of theplastid-localized 5-enolpyruvyl-3-phosphoshikimic acid synthase enzyme,also known as EPSP synthase or EPSPS, an enzyme generally thought to beunique to plants, bacteria and fungi. Recombinant plants have beentransformed with a bacterial EPSPS enzyme which is much less sensitiveto glyphosate inhibition. Therefore, plants expressing this bacterialEPSPS are less sensitive to glyphosate, and are often characterized asbeing glyphosate tolerant. Therefore, greater amounts of glyphosate canbe applied to such recombinant plants, ensuring the demise of plantswhich are susceptible or sensitive to the herbicide. However, othergenes have been identified which, when transformed into a plant genome,encoding enzymes which also provide glyphosate tolerance. One suchenzyme has been described as GOX, or glyphosate-oxidoreductase. GOXfunctions in providing protection to plants from the phosphonateherbicide glyphosate by catalyzing the degradation of glyphosate toaminomethyl phosphonic acid (AMPA) and glyoxylate. AMPA produced as aresult of glyphosate degradation can cause bleaching and stunted ordepressed plant growth, among other undesireable characteristics. Manyplant species are also sensitive to exogeneously applied AMPA, as wellas to endogenous AMPA produced as a result of GOX mediated glyphosateherbicide degradation. No method has been described which discloses theprotection of plants from applications of phosphonate herbicides such asAMPA.

[0012] Barry et al. (U.S. Pat. No. 5,633,435) disclose genes encodingEPSP synthase enzymes which are useful in producing transformed bacteriaand plants which are tolerant to glyphosate as a herbicide, as well asthe use of such genes as a method for selectively controlling weeds in aplanted transgenic crop field. Barry et al. (U.S. Pat. No. 5,463,175)disclose genes encoding glyphosate oxidoreductase (GOX) enzymes usefulin producing transformed bacteria and plants which degrade glyphosateherbicide as well as crop plants which are tolerant to glyphosate as aherbicide. Barry et al. (U.S. Pat. No. 5,463,175) disclosed theformation of AMPA as a product of GOX mediated glyphosatemetabolism.AMPA has been reported to be much less phytotoxic thanglyphosate for most plant species (Franz, 1985) but not for all plantspecies (Maier, 1983; Tanaka et al., 1986). Co-expression of a geneencoding a protein capable of neutralizing or metabolizing AMPA producedby glyphosate degradation would provide a substantial improvement overthe use of GOX alone. Thus, a method for overcoming sensitivity to AMPAformation as a result of glyphosate degradation, or a method forresistance to AMPA when used as a herbicide or as a selective agent inplant transformation methods, would be useful for providing enhanced orimproved herbicide tolerance in transgenic plants and in other organismssensitive to such compounds.

[0013] The use of glyphosate as a chemical gametocide has been described(U.S. Pat. No. 4,735,649). Therein, it is disclosed that glyphosate can,under optimal conditions, kill about 95% of male gametes, while leavingabout 40-60% of the female gametes capable of fertilization. Inaddition, a stunting effect was typically observed at the applicationlevels disclosed, shown by a reduction in the size of the plant and by aminor amount of chlorosis. Thus, a major drawback of using glyphosate asa gametocide, as is generally true with most gametocides, is thephytotoxic side effects resulting from lack of sufficient selectivityfor male gametes. These phytotoxic manifestations may be effectuated byAMPA production in transgenic plants expressing GOX after treatment withglyphosate. Therefore, it would be advantageous to provide a method forpreventing the stunting effect and chlorosis as side effects of usingglyphosate as a gametocide in transgenic plants expressing GOX.Furthermore, a more effective method would optimally kill more than 95%of male gametes or prevent male gametes from maturing and would leavegreater than 60% of female gametes substantially unaffected. It isbelieved that tissue specific co-expression of GOX with a transacylasegene encoding an enzyme capable of N-acylation of AMPA would achievethis goal.

[0014] It has now been discovered that the E. coli phnO gene encodes anenzyme having transacylase, acyltransferase, or Acyl-CoA transacylaseactivity in which a preferred substrate is a phosphonate displaying aterminal amine, and in particular amino-methyl-phosphonic acid (AMPA).The transfer of an acyl group from an Acyl-CoA to the free terminalamine of AMPA results in the formation of an N-acylated AMPA. Plants arenot known to acylate AMPA to any great extent, and some plants have beenshown to be sensitive to AMPA and insensitive to acyl-AMPA. Thus,expression of phnO in plants would be useful in enhancing thephosphonate herbicide tolerance, particularly when AMPA is used as aherbicide or selective agent in plant transformation, and moreparticularly when glyphosate is used as a herbicide in combination withrecombinant plants expressing a GOX gene.

SUMMARY OF THE INVENTION

[0015] Briefly therefore the present invention is directed to acomposition of matter comprising a novel class of genes which encodeproteins capable of N-acylation of phosphonate compounds and to methodsof using these genes and encoded proteins for improving plant toleranceto phosphonate herbicides. The present invention is also directed to amethod for selecting recombinant plants and microbes transformed withgenes encoding proteins which are capable of N-acylation of phosphonatecompounds, and to peptides which are capable of N-acylation of thecompound N-amino-methyl-phosphonic acid (N-AMPA) and other relatedphosphonate compounds. In addition, the present invention is alsodirected to a method for using plants transformed with transacylasegenes to prevent self-fertilization or to a method for enhancinghetero-fertilization in plants.

[0016] Among the several advantages found to be achieved by the presentinvention, therefore, may be noted the provision of producing stablytransformed herbicide tolerant recombinant plants which have insertedinto their genomes a polynucleotide sequence encoding a desired geneproduct, preferably an N-acyl-transferase enzyme. The polynucleotidesequence preferably is composed of a cassette containing a promotersequence which is functional in plants and which is operably linked 5′to a structural DNA sequence which, when transcribed into an RNAsequence, encodes an N-acyl-transferase enzyme peptide. The promotersequence can be heterologous with respect to the structural DNA sequenceand causes sufficient expression of the transferase enzyme in planttissue to provide herbicide tolerance to the plant transformed with thepolynucleotide sequence. The structural sequence is preferably operablylinked 3′ to a 3′ non-translated polyadenylation sequence whichfunctions in plants, and which when transcribed into RNA along with thestructural sequence causes the addition of a polyadenylated nucleotidesequence to the 3′ end of the transcribed RNA. Expression of thestructural DNA sequence produces sufficient levels of theacyltransferase enzyme in the plant tissue to enhance the herbicidetolerance of the transformed plant.

[0017] As a further embodiment, the structural DNA sequence may alsocontain an additional 5′ sequence encoding an amino-terminal peptidesequence which functions in plants to target the peptide produced fromtranslation of the structural sequence to an intracellular organelle.This additional coding sequence is preferably linked in-frame to thestructural sequence encoding the acyltransferase enzyme. The aminoterminal peptide sequence can be either a signal peptide or a transitpeptide. The intracellular organelle can be a chloroplast, amitochondrion, a vacuole, endoplasmic reticulum, or other suchstructure. The structural DNA sequence may also be linked to 5′sequences such as untranslated leader sequences (UTL's), intronsequences, or combinations of these sequences and the like which mayserve to enhance expression of the desired gene product. Intronsequences may also be introduced within the structural DNA sequenceencoding the acyltransferase enzyme. Alternatively, chloroplast orplastid transformation can result in localization of an acyltransferasecoding sequence and enzyme to the chloroplast or plastid, obviating therequirement for nuclear genome transformation, expression from thenuclear genome, and subsequent targeting of the gene product to asubcellular organelle.

[0018] Preferably, the recombinant plant expresses a gene encoding anenzyme which catalyzes the formation of AMPA. AMPA formation can resultfrom the metabolism of a naturally occurring precursor, from a precursorsuch as glyphosate provided to the plant, or can result from theformation of AMPA through some catabolic pathway. Co-expression of GOXalong with AMPA acyltransferase expression provides a plant which issurprisingly more resistant to certain phosphonate herbicides. However,one embodiment allowing plants transformed with only anN-acyltransferase to grow in the presence of AMPA or similar or relatedcompounds would provide a useful selective method for identifyinggenetically transformed plants, callus, or embryogenic tissues.

[0019] In accordance with another aspect of the present invention is theprovision of a method for selectively enhancing or improving herbicidetolerance in a recombinant plant which has inserted into its nuclear,chloroplast, plastid or mitochondrial genome a cassette comprised of apolynucleotide sequence which encodes an N-acyl-transferase enzyme.

[0020] A further embodiment encompasses the improvement of a method forselectively enhancing herbicide tolerance in a transformed plantexpressing a GOX gene which encodes a glyphosate oxidoreductase enzymeexpressed in the same plants in which an acyltransferase enzyme isproduced.

[0021] In accordance with another aspect of the present invention is theprovision of a method for producing a genetically transformed herbicidetolerant plant by inserting into a genome of a plant cell a cassettecomprising a polynucleotide sequence which encodes an N-acyl-transferaseenzyme.

[0022] A further embodiment encompasses the improvement of a method forproducing a genetically transformed herbicide tolerant plant from aplant cell expressing a GOX gene which encodes a glyphosateoxidoreductase enzyme expressed in the same plant cell in which anacyltransferase enzyme is produced.

[0023] In any of the foregoing embodiments, the herbicide tolerant plantor plant cell can be selected from the group consisting of corn, wheat,cotton, rice, soybean, sugarbeet, canola, flax, barley, oilseed rape,sunflower, potato, tobacco, tomato, alfalfa, lettuce, apple, poplar,pine, eucalyptus, acacia, poplar, sweetgum, radiata pine, loblolly pine,spruce, teak, alfalfa, clovers and other forage crops, turf grasses,oilpalm, sugarcane, banana, coffee, tea, cacao, apples, walnuts,almonds, grapes, peanuts, pulses, petunia, marigolds, vinca, begonias,geraniums, pansy, impatiens, oats, sorghum, and millet.

[0024] In accordance with another aspect of the present invention is theprovision of a peptide capable of N-acylation of the compoundN-aminomethylphosphonic acid (N-AMPA or AMPA) or other such compoundswhich are capable of causing phytotoxic effects when applied to,introduced into, or produced by plant metabolisms. One such peptide isN-aminomethylphosphonic acid transacylase (AAT) derived from expressionof an E. coli phnO structural gene sequence. Other peptides similar instructure and function to the E. coli phnO gene product are alsocontemplated.

[0025] Another aspect of the present invention is the provision of amethod for selecting cells transformed with a vector containing anacyltransferase gene expressing an enzyme capable of N-acylation of AMPAand like compounds. The method includes the steps of transforming apopulation of cells with the vector, and isolating and purifying thetransformed cells from non-transformed cells in the population afterselecting for the transformed cells by incubation in the presence ofamounts of AMPA sufficient to be inhibitory to the growth or viabilityof any non-transformed cells. The transformed cells can be bacterial,plant or fungal cells. Bacterial cells can be members of any of thefamilies encompassed by Enterobacteraceae, Mycobacteraceae,Agrobacteraceae, and Actinobacteraceae, among others. Fungal cells canbe members of Ascomycota, Basidiomycota, etc. Plant cells can be derivedfrom any member of the Plantae family.

[0026] A further embodiment of the present invention provides for amethod for producing a plant from a tissue, a cell, or other part of aplant which was derived from a plant transformed with an acyltransferasegene, a phnO gene, a gox gene, a gene in which GOX and acyltransferasepeptides are produced from a translational fusion or a transcriptionalfusion, or a polycistronic gene which encodes GOX and acyltransferasepeptides.

[0027] A further embodiment of the present invention provides for amethod for producing plants which express all or a portion of a phnOgene or similar acyltransferase gene, or a GOX gene as an antisense genein a tissue specific manner.

[0028] Other aspects also include reagents such as antibodies directedto AMPA acyltransferase, and polynucleotides for use in identifyingacyltransferase gene sequences. These reagents can be included in kitscontaining AMPA acyltransferase, polynucleotides which are or arecomplimentary to an AMPA acyltransferase gene sequence, polynucleotidesfor use in thermal amplification of an AMPA acyltransferase genesequence, antibodies directed to AMPA acyltransferase for the detectionof AMPA acyltransferase in the laboratory or in the field, and any otherreagents necessary for use in kit form as well as for use in otherassays contemplated herein.

[0029] A further object of the present invention is to provide a methodfor using phosphonate herbicides as chemical hybridizing agents. Themethod allows for selective gametocidal effects and for the productionof male sterile plants. Such plants may be engineered so that gox orphnO, or gox and phnO fail to be expressed in plant tissues required forreproduction, causing sensitivity to applied phytotoxic compounds whichinhibit formation of mature gamete structures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030]FIG. 1 illustrates a [¹⁴C] isotope detection HPLC chromatogramrepresenting a sample of a dosing solution containing only [¹⁴C]glyphosate (11.3 minutes, 98.8%), and trace amounts of [¹⁴C]AMPA (5.8minutes, 0.16%) and an unidentified [¹⁴C] material (10.2 minutes, 1%).

[0031]FIG. 2 illustrates an HPLC profile of a mixture of standards ofthe observed radioactive metabolites [¹⁴C] AMPA, [¹⁴C] glyphosate, andN-acetyl-[¹⁴C]-AMPA, as well as the impurity identified asN-acetyl-N-methyl-[¹⁴C]-AMPA.

[0032]FIG. 3 illustrates a representative HPLC profile of an extractfrom a corn callus tissue transformed with GOX and AMPAacetyltransferase, and treated with [¹⁴C] glyphosate. The peaks indicate[¹⁴C] glyphosate (10.8 minutes, 92.5% of total observed [¹⁴C]), [¹⁴C]AMPA primarily generated by GOX mediated glyphosate degradation (5.98minutes, 1.71% of total observed [¹⁴C]), and N-acetyl-[¹⁴C]AMPA producedfrom acylation of [¹⁴C] AMPA mediated by recombinant AMPAacyltransferase expressed within callus tissue (13.29 minutes, 4.54%total observed [¹⁴C]).

[0033]FIG. 4 illustrates plasmid pMON17261.

[0034]FIG. 5 illustrates plasmid pMON32571.

[0035]FIG. 6 illustrates plasmid pMON32936.

[0036]FIG. 7 illustrates plasmid pMON32946.

[0037]FIG. 8 illustrates plasmid pMON32948.

DETAILED DESCRIPTION OF THE INVENTION

[0038] The following detailed description of the invention is providedto aid those skilled in the art in practicing the present invention.Even so, the following detailed description should not be construed tounduly limit the present invention as modifications and variations inthe embodiments discussed herein may be made by those of ordinary skillin the art without departing from the spirit or scope of the presentinventive discovery.

[0039] Many words and phrases are well known in the art of molecularbiology, microbiology, protein chemistry, and plant sciences andgenerally have their plain and ordinarily understood meaning, otherwiseto be taken in context. However, the following words and phrases as usedherein have the meanings generally set forth below.

[0040] AMPA acyltransferase. As used herein, AMPA acyltransferase refersto an enzyme which functions in transferring an acyl chemical group froman acylcarrier compound such as coenzyme A, which is well known andabbreviated in the biological and chemical arts as CoA. In particular,an AMPA acyltransferase transfers an acyl chemical group from anacylcarrier to the free amino group of aminomethylphosphonate, wellknown to be a byproduct of glyphosate oxidoreductase mediated glyphosatemetabolism. AMPA acyltransferase (AAT), which herein may also be knownas AMPA acetyltransferase, AMPA transacylase, or acetyl-AMPA synthase(AAS), has been shown herein to be capable of acetyl transferaseactivity, propionyl transferase activity, malonyl transferase activity,and succinyl transferase activity. Thus, any biologically functionalequivalent of these compounds (acetyl, propionyl, malonyl, or succinyl)which serves as an acyl-carrier form of substrate capable of functioningwith an AMPA acyltransferase enzyme is within the scope of the presentinvention. One AMPA acyltranferase which has been identified, and shownby example herein to function according to the description containedherein, has previously been referred to in the art as PhnO, a proteinencoded by the phnO gene within the E. coli phn operon.

[0041] Biological functional equivalents. As used herein suchequivalents with respect to the AMPA-acyltransferase proteins of thepresent invention are peptides, polypeptides and proteins that contain asequence or moiety exhibiting sequence similarity to the novel peptidesof the present invention, such as PhnO, and which exhibit the same orsimilar functional properties as that of the polypeptides disclosedherein, including transacylase activity. Biological equivalents alsoinclude peptides, polypeptides and proteins that react with, i.e.specifically bind to antibodies raised against PhnO and that exhibit thesame or similar transacylase activity, including both monoclonal andpolyclonal antibodies.

[0042] Biological functional equivalents as used herein with respect togenes encoding acyltransferases are polynucleotides which react with thepolynucleotide sequences contemplated and described herein, i.e. whichare capable of hybridizing to a polynucleotide sequence which is or iscomplementary to a polynucleotide encoding an acyltransferase whichfunctions in transacylation of AMPA or which encode substantiallysimilar acyltransferase proteins contemplated and described herein. Aprotein which is substantially similar to the proteins described hereinis a biological functional equivalent and exhibits the same or similarfunctional properties as that of the polypeptides disclosed herein,including improved herbicide tolerance or improved herbicide resistance.Biological equivalent peptides contain a sequence or moiety such as oneor more active sites which exhibit sequence similarity to the novelpeptides of the present invention, such as PhnO. Biological equivalentsalso include peptides, polypeptides, and proteins that react with, i.e.which specifically bind to antibodies raised against PhnO and PhnO-likepeptide sequences and which exhibit the same or similar improvement inherbicidal tolerance or resistance, including both monoclonal andpolyclonal antibodies.

[0043] Chloroplast or plastid localized, as used herein, refers to abiological molecule, either polynucleotide or polypeptide, which ispositioned within the chloroplast or plastid such that the molecule isisolated from the cellular cytoplasmic milieu, and functions within thechloroplast or plastid cytoplasm to provide the effects claimed in theinstant invention. Localization of a biological molecule to thechloroplast or plastid can occur, with reference to polynucleotides, byartificial mechanical means such as electroporation, mechanicalmicroinjection, or by polynucleotide coated microprojectile bombardment,or with reference to polypeptides, by secretory or import means whereina natural, non-naturally occurring, or heterologous plastid orchloroplast targeting peptide sequence is used which functions totarget, insert, assist, or localize a linked polypeptide into achloroplast or plastid.

[0044] Event refers to a transgenic plant or plant tissue derived fromthe insertion of foreign DNA into one or more unique sites in thenuclear, mitochondrial, plastid or chloroplast DNA.

[0045] Expression: The combination of intracellular processes, includingtranscription, translation, and other intracellular protein and RNAprocessing and stabilization functions, which a coding DNA molecule suchas a structural gene is subjected to in order to produce a gene product.

[0046] Non-naturally occurring gene: A non-naturally occurringacyl-transferase gene of the present invention contains geneticinformation encoding a plant functional RNA sequence, but preferably isa gene encoding an acyl-transferase protein, whether naturally occurringor a variant of a naturally occurring protein, prepared in a mannerinvolving any sort of genetic isolation or manipulation. This includesisolation of the gene from its naturally occurring state, manipulationof the gene as by codon modification, site specific mutagenesis,truncation, introduction or removal of restriction endonuclease cleavagesites, synthesis or resynthesis of a naturally occurring sequenceencoding an acyltransferase of the present invention by in vitromethodologies such as phosphoramidite chemical synthesis methods, etc.,thermal amplification methods such as polymerase chain reaction, ligasechain reaction, inverted polymerase reaction, and the like etc., and anyother manipulative or isolative method.

[0047] Operably Linked: Nucleic acid segments connected in frame so thatthe properties of one influence the expression of the other. Forexample, a promoter sequence having properties of polymerase loading,binding, and initiation of transcription functions influences theexpression of sequences which are linked to the promoter.

[0048] Plant-Expressible Coding Regions: Coding regions which areexpressible, i.e can be transcribed and/or translated in planta, becausethey contain typical plant regulatory elements to facilitate theexpression of a gene of interest.

[0049] Plastid Transit Peptide: Any amino acid sequence useful intargeting or localizing a linked amino acid, such as a protein fusion,to a subcellular compartment or organelle such as a plastid orchloroplast. Amino acid sequences which facilitate entry into amitochondria are not altogether unlike or dissimilar from plastidtransit peptides, and are also described as transit peptides, but failto function for targeting peptide sequences to plastid or chloroplastorganelles.

[0050] Progeny of a transgenic plant includes any offspring ordescendant of the transgenic plant which contains at least oneheterologous or trans-gene, or any subsequent plant derived from thetransgenic plant which has the transgene in its lineage. Progeny is notlimited to one generation, but rather encompasses the descendants of thetransgenic plant so long as they contain or express the desiredtransgene. Seeds containing transgenic embryos as well as seeds from thetransgenic plants and their offspring or descendants are also importantparts of the invention. Transgenic cells, tissues, seeds or plants whichcontain a desired transgene are progeny of the original transgeniccells, tissue, or plant.

[0051] Promoter: A recognition site on a DNA sequence or group of DNAsequences that provides an expression control element for a structuralgene and to which RNA polymerase specifically binds and initiates RNAsynthesis (transcription) of that gene.

[0052] R₀ is the primary regenerant plant derived from transformation ofplant tissue or cells in culture. Subsequent progeny or generationsderived from the R₀ are referred to as R₁ (first generation), R₂ (secondgeneration), etc.

[0053] Regeneration: The process of producing a whole plant by growing aplant from a plant cell or plant tissue (e.g., plant protoplast orexplant).

[0054] Structural Coding Sequence refers to a DNA sequence that encodesa peptide, polypeptide, or protein that is produced followingtranscription of the structural coding sequence to messenger RNA (mRNA),followed by translation of the mRNA to produce the desired peptide,polypeptide, or protein product.

[0055] Structural gene: A gene that is expressed to produce apolypeptide.

[0056] Substantial homology: As this term is used herein, substantialhomology refers to nucleic acid sequences which are from about 40 toabout 65 percent homologous, from about 66 percent homologous to about75 percent homologous, from about 76 percent homologous to about 86percent homologous, from about 87 percent homologous to about 90 percenthomologous, from about 91 percent homologous to about 95 percenthomologous, and from about 96 percent homologous to about 99 percenthomologous to a reference polynucleotide sequence, such as either an E.coli phnO gene sequence. A first polynucleotide molecule which issubstantially homologous to a second polynucleotide molecule is or iscomplimentary to the second polynucleotide such that the firstpolynucleotide molecule hybridizes to the second polynucleotide moleculeor its complementary sequence under stringent hybridization conditions,with stringency being defined as the optimum concentration of salt andtemperature required to bring about hybridization of a firstpolynucleotide to a second polynucleotide. Methods for varyingstringency are well known in the art but may be referenced in Sambrooket al., Eds., Molecular Cloning: A Laboratory Manual, Second Edition,1989, Cold Spring Harbor Press; or Ausubel et al, Eds., Short Protocolsin Molecular Biology, Third Edition, 1995, John Wiley and Sons, Inc.Polypeptides which are believed to be within the scope if the presentinvention are those which are from about 40 to about 65 percent similar,from about 66 percent similar to about 75 percent similar, from about 76percent similar to about 86 percent similar, from about 87 percentsimilar to about 90 percent similar, from about 91 percent similar toabout 95 percent similar, and from about 96 percent similar to about 99percent similar to a reference polypeptide sequence, preferably to an E.coli PhnO peptide sequence.

[0057] Terminator: As used herein with respect to plant specificsequences intended for in planta expression, the 3′ end transcriptiontermination and polyadenylation sequence.

[0058] Transformation is a process of introducing an exogenouspolynucleotide sequence, such as a plasmid or viral vector or arecombinant polynucleotide molecule, into a cell, protoplast, plastid orchloroplast, or mitochondria in which the exogenous polynucleotidesequence is either incorporated into an endogenous polynucleotidesequence contained within the cell, or is capable of autonomousreplication. A transformed cell is a cell which has been altered by theintroduction of one or more exogenous polynucleotide molecules into thatcell. A stably transformed cell is a transformed cell which hasincorporated all or a portion of the exogenous polynucleotide into thecells' nuclear, mitochondrial, or plastid or chloroplast genomicmaterial such that the exogenous polynucleotide confers some genotypicor phenotypic trait or traits to that cell and to the progeny of thetransformed cell, measured by the detection of the exogenouslyintroduced polynucleotide, the mRNA or protein product of the exogenouspolynucleotide, a metabolite not normally produced by or found withinthe cell in the absence of the exogenous polynucleotide, or a visualinspection of the cell, plant tissue, or plants derived from thetransformed cell.

[0059] Transgene: A transgene is a polynucleotide sequence which hasbeen transferred to a cell and comprises an expression cassettecontaining a structural gene sequence encoding a desired polypeptide.The transgene is capable of being expressed when in a recipienttransformed cell, tissue, or organism. This may include an entireplasmid or other vector, or may simply include the plant functionalcoding sequence of the transferred polynucleotide. A transgenic cell isany cell derived from or regenerated from a transformed cell, includingthe initially transformed cell. Exemplary transgenic cells include plantcallus tissue derived from a transformed plant cell and particular cellssuch as leaf, root, stem, meristem, and other somatic tissue cells, orreproductive or germ line and tapetal cells obtained from a stablytransformed transgenic plant. A transgenic event is a plant or progenythereof derived from the insertion of at least one exogenouspolynucleotide into the nuclear, plastidic, or mitochondrial genome of aplant cell or protoplast. A transgenic plant is a plant or a progenythereof which has been genetically modified to contain and expressheterologous polynucleotide sequences as proteins or as RNA or DNAmolecules not previously a part of the plant composition. Asspecifically exemplified herein, a transgenic cotton plant, for example,is genetically modified to contain and express at least one heterologousDNA sequence operably linked to and under the regulatory control oftranscriptional and translational control sequences which function inplant cells or tissue or in whole plants. A transgenic plant may also bereferred to as a transformed plant. A transgenic plant also refers toprogeny of the initial transgenic plant where those progeny contain andare capable of expressing the heterologous coding sequence under theregulatory control of the plant expressible transcriptional andtranslational control sequences described herein. A transgenic plant canproduce transgenic flowers, seeds, bulbs, roots, tubers, fruit, andpollen and the like and can be crossed by conventional breeding meanswith compatible lines of plants to produce hybrid transgenic plants.

[0060] Vector: A DNA or other polynucleotide molecule capable ofreplication in a host cell and/or to which another DNA or otherpolynucleotide sequence can be operatively linked so as to bring aboutreplication of the linked sequence. A plasmid is an exemplary vector.

[0061] In accordance with the present invention, it has been discoveredthat plants can produce a phytotoxic compound when transformed withcertain genes encoding enzymes capable of degrading glyphosate. Inparticular, glyphosate oxidoreductase (GOX) mediated metabolism ofglyphosate produces a phytotoxic compound identified asN-aminomethyl-phosphonate (AMPA). Other studies have shown that anN-acylated derivative of AMPA, N-acyl-aminomethyl-phosphonate(N-acyl-AMPA or acyl-AMPA), is much less phytotoxic to most plantspecies. Enzymes have been identified which are able to covalentlymodify AMPA through an acylation mechanism, resulting in the formationof N-acyl-AMPA. One enzyme in particular causes exogeneously appliedAMPA to be N-acetylated. In plants expressing this enzyme along withGOX, phytotoxic AMPA effects are not observed.

[0062] The inventions contemplated herein take advantage of recombinantpolynucleotide cassettes comprised of elements for regulating geneexpression into which sequences, such as structural genes encodinguseful proteins, can be inserted. Insertion of such sequences into anexpression cassette is preferably accomplished using restrictionendonucleases well known in the art, however other methods for insertionare known. For example, site specific recombination methods areeffective for inserting desired sequences into such expressioncassettes. Expression cassettes contain at least a plant operablepromoter for use in initiating the production of a messenger RNAmolecule from which the useful protein is translated. Cassettes alsocontain plant operable sequences, identified as 3′ sequences, whichfunction in terminating transcription and provide untranslated sequenceswhich are 3′ polyadenylated. Thus, an expression cassette intended foruse in plants should contain at least a promoter sequence linked at its3′ end to a 3′ transcription termination and polyadenylation sequence.Preferably, a polycloning sequence or linker sequence containing one ormore unique restriction endonuclease cleavage sites is present bridgingthe promoter and 3′ sequence for convenient insertion of structural genesequences and other elements. An expression cassette intended for use inplants also preferably contains a 5′ untranslated sequence insertedbetween the promoter and the 3′ sequence. 5′ untranslated sequences(UTL's) have been shown to enhance gene expression in plants. Intronsare also contemplated as sequences which may be present in suchexpression cassettes of the present invention. The presence of plantoperable introns has also been shown, in maize in particular, to enhancegene expression in certain plant species. Introns may be present in anexpression cassette in any number of positions along the sequence of thecassette. This can include positions between the promoter and the 3′termination sequence and/or within a structural gene. There may be morethan one intron present in an expression cassette, however for thepurposes of the contemplated inventions herein, it is preferred thatintrons be present when expression cassettes are used inmonocotyledonous plants and plant tissues. Enhancer sequences are alsowell known in the art and may be present, although not necessarily as apart of an expression cassette, as enhancer sequences are known tofunction when present upstream or downstream or even at great distancesfrom a promoter driving expression of a gene of interest.

[0063] The expression of a gene localized to the plant nuclear genomeand which exists in double-stranded DNA form involves transcription toproduce a primary messenger RNA transcript (mRNA) from one strand of theDNA by RNA polymerase enzyme, and the subsequent processing of the mRNAprimary transcript inside the nucleus. This processing involves a 3′non-translated polynucleotide sequence which adds polyadenylatenucleotides to the 3′ end of the RNA. Transcription of DNA into mRNA isregulated by a sequence of DNA usually referred to as the “promoter”.The promoter comprises a sequence of bases that signals RNA polymeraseto associate with the DNA and to initiate the transcription of mRNAusing the template DNA strand to make a corresponding complementarystrand of RNA.

[0064] Those skilled in the art will recognize that there are a numberof promoters which are active in plant cells, and have been described inthe literature. Such promoters may be obtained from plants, plantviruses, or plant commensal, saprophytic, symbiotic, or pathogenicmicrobes and include, but are not limited to, the nopaline synthase(NOS) and octopine synthase (OCS) promoters (which are carried ontumor-inducing plasmids of Agrobacterium tumefaciens), the cauliflowermosaic virus (CaMV) 19S and 35S promoters, the light-inducible promoterfrom the small subunit of ribulose 1,5-bisphosphate carboxylase(ssRUBISCO, a very abundant plant polypeptide), the rice Act1 promoter,the Figwort Mosaic Virus (FMV) 35S promoter, the sugar cane bacilliformDNA virus promoter, the ubiquitin promoter, the peanut chlorotic streakvirus promoter, the comalina yellow virus promoter, the chlorophyll a/bbinding protein promoter, and meristem enhanced promoters Act2, Act8,Act11 and EF1a and the like. All of these promoters have been used tocreate various types of DNA constructs which have been expressed inplants (see e.g., McElroy et al., 1990; Barry and Kishore, U.S. Pat. No.5,463,175) and which are within the scope of the present invention.Chloroplast and plastid specific promoters, chloroplast or plastidfunctional promoters, and chloroplast or plastid operable promoters arealso envisioned. It is preferred that the particular promoter selectedshould be capable of causing sufficient in-planta expression to resultin the production of an effective amount of acyltransferase to render aplant substantially tolerant to phosphonate herbicides and products ofphosphonate herbicide metabolism. The amount of acyltransferase requiredto provide the desired tolerance may vary with the plant species.

[0065] One set of preferred promoters are constitutive promoters such asthe CaMV35S or FMV35S promoters that yield high levels of expression inmost plant organs. Enhanced or duplicated versions of the CaMV35S andFMV35S promoters are particularly useful in the practice of thisinvention (Kay et al, 1987; Rogers, U.S. Pat. No. 5,378,619). Inaddition, it may also be preferred to bring about expression of theacyltransferase gene in specific tissues of the plant, such as leaf,stem, root, tuber, seed, fruit, etc., and the promoter chosen shouldhave the desired tissue and developmental specificity. Therefore,promoter function should be optimized by selecting a promoter with thedesired tissue expression capabilities and approximate promoter strengthand selecting a transformant which produces the desired herbicidetolerance in the target tissues. This selection approach from the poolof transformants is routinely employed in expression of heterologousstructural genes in plants since there is variation betweentransformants containing the same heterologous gene due to the site ofgene insertion within the plant genome. (Commonly referred to as“position effect”). In addition to promoters which are known to causetranscription (constitutive or tissue-specific) of DNA in plant cells,other promoters may be identified for use in the current invention byscreening a plant cDNA library for genes which are selectively orpreferably expressed in the target tissues and then determine thepromoter regions.

[0066] It is preferred that the promoters utilized have relatively highexpression in all meristematic tissues in addition to other tissuesinasmuch as it is now known that phosphonate herbicides can betranslocated and accumulated in this type of plant tissue.Alternatively, a combination of chimeric genes can be used tocumulatively result in the necessary overall expression level ofacyltransferase enzyme to result in the herbicide tolerant phenotype. Apromoter which provides relatively high levels of expression can causethe production of a desired protein to in planta levels ranging from 0.1milligrams per fresh weight gram of plant tissue, to 0.5 milligrams perfresh weight gram of plant tissue, to 1.0 milligrams per fresh weightgram of plant tissue, to 2.0 or more milligrams per fresh weight gram ofplant tissue. The in planta levels of a desired protein in geneticallyisogenic crops in a field can range across a spectrum, but generally thelevels fall within 70% of a mean, more preferably within 50% of a mean,and even more preferably within 25% of a mean for all plants analyzed ina given sample.

[0067] The promoters used in the DNA constructs (i.e. chimeric plantgenes) of the present invention may be modified, if desired, to affecttheir control characteristics. For example, the CaMV35S promoter may beligated to the portion of the Arabidopsis thalianaribulose-1,5-bisphosphate carboxylase small subunit gene (ssRUBISCO)that represses the expression of ssRUBISCO in the absence of light, tocreate a promoter which is active in leaves but not in roots. Theresulting chimeric promoter may be used as described herein. Forpurposes of this description, the phrase “CaMV35S” promoter thusincludes variations of CaMV35S promoter, e.g., promoters derived bymeans of ligation with operator regions, random or controlledmutagenesis, et cetera. Furthermore, the promoters may be altered tocontain multiple “enhancer sequences” to assist in elevating geneexpression. Examples of such enhancer sequences have been reported byKay et al. (1987).

[0068] One RNA produced by a DNA construct of the present invention alsocontains a 5′ non-translated leader sequence. This sequence can bederived from the promoter selected to express the gene, and can bespecifically modified so as to increase translation of the mRNA. Thenontranslated or 5′ untranslated leader sequence (NTR or UTR) can bederived from an unrelated promoter or coding sequence. For example, the5′ non-translated regions can also be obtained from viral RNA's, fromsuitable eucaryotic genes, or from a synthetic gene sequence. Thepresent invention is not limited to constructs, as presented in one ofthe following examples, wherein the non-translated region is derivedfrom the 5′ non-translated sequence that accompanies the promotersequence. Examples of plant gene leader sequences which are useful inthe present invention are the wheat chlorophyll a/b binding protein(cab) leader and the petunia heat shock protein 70 (hsp70) leader(Winter et al., 1988).

[0069] For optimal expression in monocotyledonous plants, an intronshould also be included in the DNA expression construct. This intronwould typically be placed near the 5′ end of the mRNA in untranslatedsequence. This intron could be obtained from, but not limited to, a setof introns consisting of the maize hsp70 intron (Brown et al., U.S. Pat.No. 5,424,412; 1995) or the rice Act1 intron (McElroy et al., 1990).

[0070] Where more than one expression cassette in included within aplasmid or other polynucleotide construct, a first expression cassettecomprising a DNA molecule typically contains a constitutive promoter, astructural DNA sequence encoding a glyphosate oxidoreductase enzyme(GOX), and a 3′ non-translated region. A second expression cassettecomprising a DNA molecule typically contains a constitutive promoter, astructural DNA sequence encoding an N-acyl-transferase enzyme which iscapable of reacting with AMPA to produce N-acyl-AMPA, and a 3′non-translated region. Additional expression cassettes comprising a DNAmolecule are also envisioned. For example, genes encoding insecticidalor fungicidal activities, drought or heat tolerance, antibioticcompounds, pharmaceutical compounds or reagents such as tumor suppressorproteins or antibody components, biopolymers, other commercially usefulcompounds and the like may also be expressed in the plants envisioned bythe present invention, along with genes which provide increasedherbicide tolerance. A number of constitutive promoters which are activein plant cells have been described. Suitable promoters for constitutiveexpression of either GOX or an N-acyl-transferase include, but are notlimited to, the cauliflower mosaic virus (CaMV) 35S promoter (Odell etal. 1985), the Figwort mosaic virus (FMV) 35S (Sanger et al. 1990), thesugarcane bacilliform DNA virus promoter (Bouhida et al., 1993), thecommelina yellow mottle virus promoter (Medberry and Olszewski 1993),the light-inducible promoter from the small subunit of theribulose-1,5-bis-phosphate carboxylase (ssRUBISCO) (Coruzzi et al.,1984), the rice cytosolic triosephosphate isomerase (TPI) promoter (Xuet al. 1994), the adenine phosphoribosyltransferase (APRT) promoter ofArabidopsis (Moffatt et al. 1994), the rice actin 1 gene promoter (Zhonget al. 1996), and the mannopine synthase and octopine synthase promoters(Ni et al. 1995). All of these promoters have been used to createvarious types of plant-expressible recombinant DNA constructs.Comparative analysis of constitutive promoters by the expression ofreporter genes such as the uidA (β-glucuronidase) gene from E. coli hasbeen performed with many of these and other promoters ( Li et al. 1997;Wen et al. 1993).

[0071] Promoters used in the second cassette comprising a DNA moleculecan be selected to control or limit specific expression where celllethality is desired. In a preferred embodiment, the promoter will becapable of directing expression exclusively or primarily in tissuescritical for plant survival or plant viability, while limitingexpression of the second cassette comprising a DNA molecule in othernonessential tissues. For example, tissues which differentiate intopollen development or terminal tissues such as the pollen itself, thetapetal cell layer of the anther, or the anther tissues. Alternatively,plant promoters capable of regulating the expression of genes inparticular cell and tissue types are well known. Those that are mostpreferred in the embodiments of this invention are promoters whichexpress specifically during the development of the male reproductivetissue or in pollen at levels sufficient to produce inhibitory RNAmolecules complementary to the sense RNA transcribed by the constitutivepromoter of the first expression cassette comprising a DNA molecule.Examples of these types of promoters include the TA29 tobaccotapetum-specific promoter (Mariani et al. 1990), the PA1 and PA2chalcone flavonone isomerase promoters from petunia (van Tunen et al.1990), the SLG gene promoter from Brassica oleracea (Heizmann et al.1991), and LAT gene promoters from tomato (Twell et al. 1991).

[0072] Anther and pollen-specific promoters from rice have beenisolated. Examples include the Osg6B promoter, which was shown to driveexpression of the β-glucuronidase gene in transgenic rice in immatureanthers. No activity was detected in other tissues of spikelets, leavesor roots (Yokoi et al. 1997). The PS1 pollen-specific promoter from ricehas been shown to specifically express the β-glucuronidase gene in ricepollen (Zou et al. 1994). Additional rice genes have been identifiedthat specifically express in the anther tapetum of rice (Tsuchiya et al.1994, Tsuchiya et al. 1997). The isolation of additional genes expressedpredominantly during anther development in rice can be performed, forexample, by construction of a cDNA library to identify anther specificclones (Qu et al.).

[0073] Those skilled in the art are aware of the approaches used in theisolation of promoters which function in plants, and from genes ormembers of gene families that are highly expressed in particular planttissues such as in roots, shoots, meristem, leaves, flowers, fruits, inpollen, or in plant cell types involved in the production of pollen(Stinson et al. 1987; Brown and Crouch. 1990; McCormick et al. 1989).Further examples of tissue specific promoters include the promoter forthe exopolygalacturonase gene of maize (Dubald, et al. 1993) and thepromoter for the Zmc13 mRNA (Hanson, et al. 1989). Promoters which havebeen shown to preferentially express in tomato pollen are the LAT52 andLAT59 promoters (Twell et al. 1991). A portion of the maize pZtappromoter sequence (psgB6-1) was disclosed in U.S. Pat. No. 5,470,359.

[0074] A recombinant DNA molecule of the present invention typicallycomprises a promoter operably or operatively linked to a DNA sequenceencoding a 5′ non-translated region, a DNA sequence of a plant intron, astructural sequence encoding a chloroplast transit peptide (CTP), a DNAcoding sequence for a gene encoding improved herbicide tolerance, and a3′ non-translated region.

[0075] The 5′ non-translated leader sequence can be derived from thepromoter selected to express the heterologous DNA sequence, and can bespecifically modified if desired so as to increase translation of mRNA.A 5′ non-translated region can also be obtained from viral RNAs, fromsuitable eukaryotic genes, or from a synthetic gene sequence. Thepresent invention is not limited to constructs wherein thenon-translated region is derived from the 5′ non-translated sequencewhich accompanies the promoter sequence. The leader sequence could alsobe derived from an unrelated promoter or coding sequence.

[0076] The 3′ non-translated region of a plant operable recombinant DNAmolecule contains a polyadenylation signal which functions in plants tocause the addition of adenylate nucleotides to the 3′ end of the RNA.The 3′ non-translated region can be obtained from various genes whichare expressed in plant cells. The nopaline synthase 3′ untranslatedregion (Fraley et al. 1983), the 3′ untranslated region from peassRUBISCO (Coruzzi et al. 1994), the 3′ untranslated region from soybean7S seed storage protein gene (Schuler et al. 1982) and the pea smallsubunit of the pea ssRUBISCO gene are commonly used in this capacity.The 3′ transcribed, non-translated regions containing the polyadenylatesignal of Agrobacterium tumor-inducing (Ti) plasmid genes are alsosuitable.

[0077] Examples of plant introns suitable for expression in monocotsincludes, for example, maize hsp70 intron, rice actin 1 intron, maizeADH 1 intron, Arabidopsis SSU intron, Arabidopsis EPSPS intron, petuniaEPSPS intron and others known to those skilled in the art.

[0078] It may be particularly advantageous to direct the localization ofproteins conferring herbicide tolerance to subcellular compartment, forexample, to the mitochondrion, endoplasmic reticulum, vacuoles,chloroplast or other plastidic compartment. Proteins can be directed tothe chloroplast by including at their amino-terminus a chloroplasttransit peptide (CTP). Naturally occurring chloroplast targetedproteins, synthesized as larger precursor proteins containing anamino-terminal chloroplast targeting peptide directing the precursor tothe chloroplast import machinery, have been previously identified andare well known in the art. Chloroplast targeting peptides are generallycleaved by specific endoproteases located within the chloroplastorganelle, thus releasing the targeted mature and preferably activeenzyme from the precursor into the chloroplast melieu. Examples ofsequences encoding peptides which are suitable for directing thetargeting of the herbicide tolerance gene or transacylase gene productto the chloroplast or plastid of the plant cell include the petuniaEPSPS CTP, the Arabidopsis EPSPS CTP2 and intron, and others known tothose skilled in the art. Such targeting sequences provide for thedesired expressed protein to be transferred to the cell structure inwhich it most effectively functions, or by transferring the desiredexpressed protein to areas of the cell in which cellular processesnecessary for desired phenotypic function are concentrated. Chloroplasttargeting peptides have been found to be particularly useful in theselection of glyphosate resistant plants (Barry et al., U.S. Pat. No.5,463,175; Barry et al., U.S. Pat. No. 5,633,435). Glyphosate functionsto kill the cell by inhibiting aromatic amino acid biosynthesis whichtakes place within the chloroplast. Therefor, concentrating theresistance gene product within the chloroplast provides increasedresistance to the herbicide. The examples herein provide for atransacylase which is also targeted to or localized to and concentratedwithin the chloroplast. Specific examples of chloroplast targetingpeptides are well known in the art and include the Arabidopsis thalianaribulose bisphosphate carboxylase small subunit ats1A transit peptide,an Arabidopsis thaliana EPSPS transit peptide, and a Zea maize ribulosebisphosphate carboxylase small subunit transit peptide. One CTP that hasfunctioned herein to localize heterologous proteins to the chloroplastwas derived from the Arabidopsis thaliana ribulose bisphosphatecarboxylase small subunit ats1A transit peptide. A polynucleotidesequence encoding a variant of this transit peptide used herein providesthe native transit peptide amino acid sequence plus a reiteration of thetransit peptide cleavage site, and has been shown herein to be usefulfor deploying active recombinant transacylase enzyme to the chloroplast(SEQ ID NO: 9).

[0079] An alternative means for localizing plant operable herbicidetolerance or herbicide resistance genes to a chloroplast or plastidincludes chloroplast or plastid transformation. Recombinant plants canbe produced in which only the mitochondrial or chloroplast DNA has beenaltered to incorporate the molecules envisioned in this application.Promoters which function in chloroplasts have been known in the art(Hanley-Bowden et al., Trends in Biochemical Sciences 12:67-70, 1987).Methods and compositions for obtaining cells containing chloroplastsinto which heterologous DNA has been inserted have been described, forexample by Daniell et al. (U.S. Pat. No. 5,693,507; 1997) and Maliga etal. (U.S. Pat. No. 5,451,513; 1995).

[0080] The accumulation of AMPA in plants can cause phytotoxic symptomswhich are manifested phenotypically as chlorosis of the leaves, stuntedgrowth, infertility, and death, although not all of these symptoms areevidenced in every species of plant. It has been discovered herein thatenzymatic modification of the AMPA molecule by transacylation to produceN-acyl-AMPA provides a means for overcoming the phytotoxic effects ofAMPA. A method for assaying the conversion of AMPA to N-acyl-AMPAinvolves providing [¹⁴C] labeled AMPA as one substrate for thetransacylase enzyme, and acyl-CoA as another substrate for the enzyme inan aqueous reaction volume, and separating the [¹⁴C] labeled AMPAsubstrate from N-acyl-[¹⁴C]-AMPA product by HPLC on an anion exchangecolumn as described in the examples herein. Surprisingly, thetransacylase enzyme has been shown to be capable of utilizing otheracylated-CoA compounds as substrates for transacylating the AMPAsubstrate. In particular, propionyl-CoA was shown to be a particularlyreactive substrate for the transacylation reaction in vitro, producingN-propionyl-[¹⁴C]-AMPA. Larger acylated-CoA compounds, i.e. butyryl-CoAor methylmalonyl-CoA and other organic molecules covalently linked toCoA which have a carbon chain length greater than C₃ proved to be lesseffective in the transacylation reaction when using AMPA as theacyl-group recipient substrate. Notwithstanding this information, oneskilled in the art would recognize that other transacylases which aresubstantially related by amino acid sequence homology to a PhnO orPhnO-like enzyme as characterized herein would have a similar substratespecificity in the AMPA transacylase reaction as compared to thatencompassed by PhnO. These other enzymes too are conceptually within thescope and spirit of the invention described herein. For example, fattyacid biosynthesis is mediated by a wide range of acyl-CoA andacyl-carrier protein compounds which may be useful as substrates intransacylating phytotoxic compounds such as AMPA. A transacylase capableof AMPA transacylation using a fatty acid intermediate could conceivablyprovide plant protection by eliminating AMPA phytotoxicity. An enzymesuch as PhnO, which is capable of transacylation, may be useful indetoxifying a wide range of toxic compounds which contain CP bonds andwhich additionally contain a CN linkage.

[0081] Methods and compositions for transforming a bacterium, a yeast orfungal cell, a plant cell, or an entire plant with one or moreexpression vectors comprising a phnO- or phnO-like gene sequence arefurther aspects of this disclosure. A transgenic bacterium, yeast orfungal cell, plant cell, or plant derived from such a transformationprocess or the progeny and seeds from such a transgenic plant are alsofurther embodiments of this invention.

[0082] Methods for transforming bacteria and yeast or fungal cells arewell known in the art. Typically, means of transformation are similar tothose well known means used to transform other bacteria, such as E.coli, or yeast, such as Saccharomyces cerevisiae. Methods for DNAtransformation of plant cells include, but are not limited toAgrobacterium-mediated plant transformation, protoplast transformation,gene transfer into pollen, injection into reproductive organs, injectioninto immature embryos, plastid or chloroplast transformation, andparticle bombardment. Each of these methods has distinct advantages anddisadvantages. Thus, one particular method of introducing genes into aparticular plant species may not be the most effective for another plantspecies, but it is well known by those skilled in the art which methodsare useful for a particular plant species.

[0083] There are many methods for introducing transforming DNA segmentsinto cells, but not all are suitable for delivering DNA to plant cells.Suitable methods are believed to include virtually any method by whichDNA can be introduced into a cell, such as by Agrobacterium infection,binary bacterial artificial chromosome (BIBAC) vectors (Hamilton et al.,1996), direct delivery of DNA such as, for example by PEG-mediatedtransformation of protoplasts (Omirulleh et al., 1993), bydesiccation/inhibition-mediated DNA uptake, by electroporation, byagitation with silicon carbide fibers, by acceleration of DNA coatedparticles, etc. In certain embodiments, acceleration methods arepreferred and include, for example, microprojectile bombardment and thelike.

[0084] Technology for introduction of DNA into cells is well-known tothose of skill in the art. Four general methods for delivering a geneinto cells have been described: (1) chemical methods (Graham and van derEb, 1973; Zatloukal et al., 1992); (2) physical methods such asmicroinjection (Capecchi, 1980), electroporation (Wong and Neuman, 1982;Fromm et al, 1985; U.S. Pat. No. 5,384,253) and the gene gun (Johnstonand Tang, 1994; Fynan et al., 1993; Luthra et al., 1997); (3) viralvectors (Clapp, 1993; Lu et al., 1993; Eglitis and Anderson, 1988a;1988b); and (4) receptor-mediated mechanisms (Curiel et al., 1991; 1992;Wagner et al., 1992)

[0085] Methods for transforming dicots, primarily by use ofAgrobacterium tumefaciens, and obtaining transgenic plants have beenpublished for cotton (U.S. Pat. No. 5,004,863; U.S. Pat. No. 5,159,135;U.S. Pat. No. 5,518,908), soybean (U.S. Pat. No. 5,569,834; U.S. Pat.No. 5,416,011; McCabe et al. (1988); Christou et al. (1988)), Brassica(U.S. Pat. No. 5,463,174), peanut (Cheng et al. (1996); De Kathen andJabobsen (1990)).

[0086] Transformation of monocots using electroporation, particlebombardment, and Agrobacterium have also been reported. Transformationand plant regeneration have been achieved in asparagus (Bytebier et al.(1987)), barley (Wan and Lemaux (1994)), maize (Rhodes et al. (1988);Ishida et al. (1996); Gordon-Kamm et al. (1990); Fromm et al. (1990);Koziel et al. (1993); Armstrong et al (1995), oat (Somers et al.(1992)), orchardgrass (Horn et al. (1988)), rice (Toriyama et al.(1988); Park et al. (1996); Abedinia et al. (1997); Zhang and Wu (1988);Zhang et al. (1988); Battraw and Hall (1990); Christou et al. (1991);Park et al. (1996)), rye (De la Pena et al. (1987)), sugar cane (Bowerand Birch (1992)), tall fescue (Wang et al. (1992)), and wheat (Vasil etal. (1992); Weeks et al (1993)). Techniques for monocot transformationand plant regeneration are also discussed in Davey et al. (1986).

[0087] Recombinant plants could also be produced in which only themitochondrial or chloroplast DNA has been altered to incorporate themolecules envisioned in this application. Promoters which function inchloroplasts have been known in the art (Handley-Bowden et al., Trendsin Biochemical Sciences 12:67-70, 1987). Methods and compositions forobtaining cells containing chloroplasts into which heterologous DNA hasbeen inserted has been described by Daniell et al., U.S. Pat. No.5,693,507 (1997) and Maliga et al. (U.S. Pat. No. 5,451,513; 1995).Recombinant plants which have been transformed using heterologous DNA,altering both nuclear and chloroplast or plastidic genomes is alsowithin the scope of this invention.

[0088] The present invention discloses DNA constructs comprisingpolynucleotide sequences encoding AMPA-transacylase. Methods foridentifying and isolating heterologous genes encoding peptides whichfunction in N-acylation of AMPA are disclosed herein. Methods for theconstruction and expression of synthetic genes in plants are well knownby those of skill in the art and are described in detail in U.S. Pat.No. 5,500,365, and in monocotyledonous plants in particular in U.S. Pat.No. 5,689,052. The present invention contemplates the use of AMPAacyltransferase genes alone or in combination with genes encoding GOXmediated glyphosate degradation enzymes in the transformation of bothmonocotyledonous and dicotyledonous plants. To potentiate the expressionof these genes, the present invention provides DNA constructs comprisingpolynucleotide sequences encoding these types of proteins which arelocalized to the plant cell cytoplasm as well as sequences encodingplastid targeting peptides positioned upstream of the polynucleotidesequences encoding the AMPA transacylase and/or GOX proteins.

[0089] In one aspect, nucleotide sequence information provided by theinvention allows for the preparation of relatively short DNA sequenceshaving the ability to specifically hybridize to gene sequences of theselected polynucleotides disclosed herein. In these aspects, nucleicacid probes of an appropriate length are prepared based on aconsideration of selected polynucleotide sequences encoding AMPAtransacylase polypeptides, e.g., sequences such as are shown in SEQ IDNO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. Such nucleic acid probes may alsobe prepared based on a consideration of selected polynucleotidesequences encoding a plastid targeting peptide, such as those shown inSEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 14. Theability of such nucleic acid probes to specifically hybridize to a genesequence encoding an AMPA transacylase polypeptide or a plastidtargeting peptide sequence lends to them particular utility in a varietyof embodiments. Most importantly, the probes may be used in a variety ofassays for detecting the presence of complementary sequences in a givensample.

[0090] In certain embodiments, it is advantageous to use oligonucleotideprimers. The sequence of such primers is designed using a polynucleotideof the present invention for use in detecting, amplifying or mutating adefined sequence of a AMPA transacylase gene from any suitable organismusing PCR™ technology. The process may also be used to detect, amplifyor mutate a defined sequence of the polynucleotide encoding a plastidtargeting peptide. Segments of genes related to the polynucleotidesencoding the AMPA transacylase polypeptides and plastid targetingpeptides of the present invention may also be amplified by PCR™ usingsuch primers.

[0091] To provide certain of the advantages in accordance with thepresent invention, a preferred nucleic acid sequence employed forhybridization studies or assays includes sequences that aresubstantially complementary to at least a length of 14 to 30 or soconsecutive nucleotides of a polynucleotide sequence flanking, in ciswith, or encoding an AMPA transacylase, such as that shown in SEQ ID NO:5 or SEQ ID NO: 6, or sequences that are substantially complementary toat least a length of 14 to 30 or so consecutive nucleotides of asequence encoding a plastid targeting peptide. By “substantiallycomplimentary”, it is meant that a polynucleotide is preferably about70% complimentary, or more preferably about 80% complimentary, or evenmore preferably about 90% complimentary, or most preferably about99-100% complimentary in sequence to a target polynucleotide sequence.

[0092] A size of at least 14 nucleotides in length helps to ensure thatthe fragment will be of sufficient length to form a duplex molecule thatis both stable and selective. Molecules having complementary sequencesover segments greater than 14 bases in length are generally preferred Inorder to increase stability and selectivity of the hybrid, and therebyimprove the quality and degree of specific hybrid molecules obtained,one will generally prefer to design nucleic acid molecules havinggene-complementary sequences of 14 to 20 nucleotides, or even longerwhere desired. Such fragments may be readily prepared by, for example,directly synthesizing the fragment by chemical means, such asphosphoramidite chemistries for example; by application of nucleic acidreproduction technology, such as the PCR™ technology of U.S. Pat. Nos.4,683,195, and 4,683,202 (each specifically incorporated herein byreference); or by excising selected DNA fragments from recombinantplasmids containing appropriate inserts and suitable restriction sites.

[0093] The present invention also contemplates an expression vectorcomprising a polynucleotide of the present invention. Thus, in oneembodiment an expression vector is an isolated and purified DNA moleculecomprising a promoter operably linked to a coding region that encodes apolypeptide of the present invention, which coding region is operativelylinked to a transcription-terminating region, whereby the promoterdrives the transcription of the coding region. The coding region mayinclude a segment or sequence encoding a AMPA transacylase and a segmentor sequence encoding a plastid targeting peptide. The DNA moleculecomprising the expression vector may also contain a plant functionalintron, and may also contain other plant functional elements such assequences encoding untranslated sequences (UTL's) and sequences whichact as enhancers of transcription or translation.

[0094] As used herein, the terms “operatively linked” or “operablylinked” mean that a sequence which functions as a promoter is connectedor linked to a coding region in such a way that the transcription ofthat coding region is controlled and regulated by that promoter. Meansfor operatively linking a promoter to a coding region to regulate bothupstream and downstream are well known in the art.

[0095] Preferred plant transformation vectors include those derived froma Ti plasmid of Agrobacterium tumefaciens, as well as those disclosed,e.g., by Herrera-Estrella (1983), Bevan (1983), Klee (1985) and Eur.Pat. Appl. No. EP 0120516 (each specifically incorporated herein byreference). In addition, plant preferred transformation vectors directedto chloroplast or plastid transformation include those disclosed in U.S.Pat. No. 5,693,507 (1997), U.S. Pat. No. 5,451,513 (1995), McBride etal. (1995), Staub et al. (1995a), Staub et al. (1995b), and WO 95/24492(each specifically incorporated herein by reference).

[0096] Where an expression vector of the present invention is to be usedto transform a plant, a promoter is selected that has the ability todrive expression in that particular species of plant. Promoters thatfunction in different plant species are also well known in the art.Promoters useful in expressing the polypeptide in plants are those whichare inducible, viral, synthetic, or constitutive as described (Odell etal., 1985), and/or temporally regulated, spatially regulated, andspatio-temporally regulated. Preferred promoters include the enhancedCaMV35S promoters, and the FMV35S promoter.

[0097] The expression of a gene which exists in double-stranded DNA formlocalized to the plant nuclear genome involves transcription ofmessenger RNA (mRNA) from the coding strand of the DNA by an RNApolymerase enzyme, and the subsequent processing of the mRNA primarytranscript inside the nucleus. Genes expressed from within a chloroplastor plastid also produce an mRNA transcript which is not processedfurther prior to translation. In any event, transcription of DNA intomRNA is regulated by a region of DNA referred to as the “promoter”. TheDNA comprising the promoter is represented by a sequence of bases thatsignals RNA polymerase to associate with the DNA and to initiate thetranscription of mRNA using one of the DNA strands as a template to makea corresponding strand of RNA. The particular promoter selected shouldbe capable of causing sufficient expression of an AMPA acyltransferaseenzyme coding sequence to result in the production of an herbicidetolerance effective or herbicide resistance effective amount of thetransacylase protein localized to the desired intracellular location.

[0098] Structural genes can be driven by a variety of promoters in planttissues. Promoters can be near-constitutive (i.e. they drivetranscription of the transgene in all tissue), such as the CaMV35Spromoter, or tissue-specific or developmentally specific promotersaffecting dicots or monocots. Where the promoter is a near-constitutivepromoter such as CaMV35S or FMV35S, increases in polypeptide expressionare found in a variety of transformed plant tissues and most plantorgans (e.g., callus, leaf, seed, stem, meristem, flower, and root).Enhanced or duplicate versions of the CaMV35S and FMV35S promoters areparticularly useful in the practice of this invention (Kay et al., 1987;Rogers, U.S. Pat. No. 5,378,619).

[0099] Those skilled in the art will recognize that there are a numberof promoters which are active in plant cells, and have been described inthe literature. Such promoters may be obtained from plants or plantviruses and include, but are not limited to, the nopaline synthase (NOS)and octopine synthase (OCS) promoters (which are carried ontumor-inducing plasmids of A. tumefaciens), the cauliflower mosaic virus(CaMV) 19S and 35S promoters, the light-inducible promoter from thesmall subunit of ribulose 1,5-bisphosphate carboxylase (ssRUBISCO, avery abundant plant polypeptide), the rice Act1 promoter and the FigwortMosaic Virus (FMV) 35S promoter. All of these promoters have been usedto create various types of DNA constructs which have been expressed inplants (see e.g., McElroy et al., 1990, U.S. Pat. No. 5,463,175).

[0100] In addition, it may also be preferred to bring about expressionof genes such as an AMPA acyltransferase which improve herbicidetolerance or herbicide resistance in specific tissues of a plant byusing plant integrating vectors containing a tissue-specific promoter.Specific target tissues may include the leaf, stem, root, tuber, seed,fruit, etc., and the promoter chosen should have the desired tissue anddevelopmental specificity. Therefore, promoter function should beoptimized by selecting a promoter with the desired tissue expressioncapabilities and approximate promoter strength, and selecting atransformant which produces the desired transacylase activity in thetarget tissues. This selection approach from the pool of transformantsis routinely employed in expression of heterologous structural genes inplants since there is variation between transformants containing thesame heterologous gene due to the site of gene insertion within theplant genome (commonly referred to as “position effect”). In addition topromoters which are known to cause transcription (constitutive ortissue-specific) of DNA in plant cells, other promoters may beidentified for use in the current invention by screening a plant cDNAlibrary for genes which are selectively or preferably expressed in thetarget tissues, then determining the promoter regions. Chloroplast orplastid functional promoters are known in the art (Hanley-Bowden et al.,Daniell et al., Maliga et al.).

[0101] Other exemplary tissue-specific promoters are corn sucrosesynthetase 1 (Yang et al., 1990), corn alcohol dehydrogenase 1 (Vogel etal., 1989), corn light harvesting complex (Simpson, 1986), corn heatshock protein (Odell et al., 1985), pea small subunit RuBP carboxylase(Poulsen et al., 1986; Cashmore et al., 1983), Ti plasmid mannopinesynthase (McBride and Summerfelt, 1989), Ti plasmid nopaline synthase(Langridge et al., 1989), petunia chalcone isomerase (Van Tunen et al.,1988), bean glycine rich protein 1 (Keller et al., 1989), CaMV 35stranscript (Odell et al., 1985) and Potato patatin (Wenzler et al, 1989)promoters. Preferred promoters are the cauliflower mosaic virus (CaMV35S) promoter and the S-E9 small subunit RuBP carboxylase promoter.

[0102] The promoters used in the DNA constructs of the present inventionmay be modified, if desired, to affect their control characteristics.For example, the CaMV35S promoter may be ligated to the portion of thessRUBISCO gene that represses the expression of ssRUBISCO in the absenceof light, to create a promoter which is active in leaves but not inroots. For purposes of this description, the phrase “CaMV35S” promoterthus includes variations of CaMV35S promoter, e.g., promoters derived bymeans of ligation with operator regions, random or controlledmutagenesis, etc. Furthermore, the promoters may be altered to containmultiple “enhancer sequences” to assist in elevating gene expression.Examples of such enhancer sequences have been reported by Kay et al.(1987). Chloroplast or plastid specific promoters are known in the art(Daniell et al., U.S. Pat. No. 5,693,507; herein incorporated byreference). Promoters obtainable from chloroplast genes, for example,such as the psbA gene from spinach or pea, the rbcL and atpB promoterregions from maize, and rRNA promoters. Any chloroplast or plastidoperable promoter is within the scope of the present invention.

[0103] A transgenic plant of the present invention produced from a plantcell transformed with a tissue specific promoter can be crossed with asecond transgenic plant developed from a plant cell transformed with adifferent tissue specific promoter to produce a hybrid transgenic plantthat shows the effects of transformation in more than one specifictissue.

[0104] The RNA produced by a DNA construct of the present invention mayalso contain a 5′ non-translated leader sequence (5′UTL). This sequencecan be derived from the promoter selected to express the gene, and canbe specifically modified so as to increase translation of the mRNA. The5′ non-translated regions can also be obtained from viral RNAs, fromsuitable eukaryotic genes, or from a synthetic gene sequence. Thepresent invention is not limited to constructs wherein thenon-translated region is derived from the 5′ non-translated sequencethat accompanies the promoter sequence. One plant gene leader sequencefor use in the present invention is the petunia heat shock protein 70(hsp70) leader (Winter et al., 1988).

[0105] 5′ UTL's are capable of regulating gene expression when localizedto the DNA sequence between the transcription initiation site and thestart of the coding sequence. Compilations of leader sequences have beenmade to predict optimum or sub-optimum sequences and generate“consensus” and preferred leader sequences (Joshi, 1987). Preferredleader sequences are contemplated to include those which comprisesequences predicted to direct optimum expression of the linkedstructural gene, i.e. to include a preferred consensus leader sequencewhich may increase or maintain mRNA stability and prevent inappropriateinitiation of translation. The choice of such sequences will be known tothose of skill in the art in light of the present disclosure. Sequencesthat are derived from genes that are highly expressed in plants, and inmaize in particular, will be most preferred. One particularly usefulleader may be the petunia HSP70 leader.

[0106] In accordance with the present invention, expression vectorsdesigned to specifically potentiate the expression of the polypeptide inthe transformed plant may include certain regions encoding plastid orchloroplast targeting peptides, herein abbreviated in various forms asCTP, CTP1, CTP2, etc., each representing a different or varianttargeting peptide sequence. These regions allow for the cellularprocesses involved in transcription, translation and expression of theencoded protein to be fully exploited when associated with certain GOXor AMPA transacylase protein sequences. Such targeting peptides functionin a variety of ways, such as for example, by transferring the expressedprotein to the cell structure in which it most effectively operates, orby transferring the expressed protein to areas of the cell in whichcellular processes necessary for expression are concentrated. The use ofCTP's may also increase the frequency of recovery of morphologicallynormal plants, and the frequency at which transgenic plants may berecovered.

[0107] Chloroplast targeting peptides have been found particularlyuseful in the glyphosate resistant selectable marker system. In thissystem, plants transformed to express a protein conferring glyphosateresistance are transformed along with a CTP that targets the peptide tothe plant cell's chloroplasts. Glyphosate inhibits the shikimic acidpathway which leads to the biosynthesis of aromatic compounds includingamino acids and vitamins. Specifically, glyphosate inhibits theconversion of phosphoenolpyruvic acid and 3-phosphoshikimic acid to5-enolpyruvyl-3-phosphoshikimic acid by inhibiting the enzyme5-enolpyruvyl-3-phosphoshikimic acid synthase (EPSP synthase or EPSPS).Introduction of a transgene encoding EPSPS allows the plant cell toresist the effects of glyphosate, especially when the transgene encodesa glyphosate insensitive EPSPS enzyme. Thus, as the herbicide glyphosatefunctions to kill the cell by interrupting aromatic amino acidbiosynthesis, particularly in the cell's chloroplast, the CTP allowsincreased resistance to the herbicide by concentrating what glyphosateresistance enzyme the cell expresses in the chloroplast, i.e. in thetarget organelle of the cell. Exemplary herbicide resistance enzymesinclude EPSPS and glyphosate oxido-reductase (GOX) genes (see Comai,1985, U.S. Pat. No. 4,535,060, specifically incorporated herein byreference in its entirety).

[0108] CTPs can target proteins to chloroplasts and other plastids. Forexample, the target organelle may be the amyloplast. Preferred CTP's ofthe present invention include those targeting both chloroplasts as wellas other plastids. Specific examples of preferred CTP's include themaize RUBISCO SSU protein CTP, and functionally related peptides such asthe Arabidopsis thaliana RUBISCO small subunit CTP and the Arabidopsisthaliana EPSPS CTP. These CTP's are exemplified by the polynucleotideand amino acid sequences shown in SEQ ID NO: 9, SEQ ID NO: 11, SEQ IDNO: 13, and SEQ ID NO: 14 respectively.

[0109] Recombinant plants, cells, seeds, and other plant tissues couldalso be produced in which only the mitochondrial or chloroplast DNA hasbeen altered to incorporate the molecules envisioned in thisapplication. Promoters which function in chloroplasts have been known inthe art (Hanley-Bowden et al., Trends in Biochemical Sciences 12:67-70,1987). Methods and compositions for obtaining cells containingchloroplasts into which heterologous DNA has been inserted has beendescribed in U.S. Pat. No. 5,693,507 (1997). McBride et al. (WO95/24492) disclose localization and expression of genes encoding Cry1Aδ-endotoxin protein in tobacco plant chloroplast genomes.

[0110] An exemplary embodiment of the invention involves the plastid orchloroplast targeting or plastid or chloroplast localization of genesencoding enzymes or proteins conferring herbicide tolerance or herbicideresistance in plants. Plastid or chloroplast targeting sequences havebeen isolated from numerous nuclear encoded plant genes and have beenshown to direct importation of cytoplasmically synthesized proteins intoplastids or chloroplasts (reviewed in Keegstra and Olsen, 1989). Avariety of plastid targeting sequences, well known in the art, includingbut not limited to ADPGPP, EPSP synthase, or ssRUBISCO, may be utilizedin practicing this invention. In addition, plastidic targeting sequences(peptide and nucleic acid) for monocotyledonous crops may consist of agenomic coding fragment containing an intron sequence as well as aduplicated proteolytic cleavage site in the encoded plastidic targetingsequences.

[0111] The preferred CTP sequence for dicotyledonous crops is referredto herein as (SEQ ID NO: 9), and consists of a genomic coding fragmentcontaining the chloroplast targeting peptide sequence from the EPSPsynthase gene of Arabidopsis thaliana in which the transit peptidecleavage site of the pea ssRUBISCO CTP replaces the native EPSP synthaseCTP cleavage site (Klee et al., 1987).

[0112] For optimized expression in monocotyledonous plants, an intronmay also be included in the DNA expression construct. Such an intron istypically placed near the 5′ end of the mRNA in untranslated sequence.This intron could be obtained from, but not limited to, a set of intronsconsisting of the maize heat shock protein (HSP) 70 intron (U.S. Pat.No. 5,424,412; 1995), the rice Act1 intron (McElroy et al., 1990), theAdh intron 1 (Callis et al., 1987), or the sucrose synthase intron(Vasil et al., 1989).

[0113] The 3′ non-translated region of the genes of the presentinvention which are localized to the plant nuclear genome also contain apolyadenylation signal which functions in plants to cause the additionof adenylate nucleotides to the 3′ end of the mRNA. RNA polymerasetranscribes a nuclear genome coding DNA sequence through a site wherepolyadenylation occurs. Typically, DNA sequences located a few hundredbase pairs downstream of the polyadenylation site serve to terminatetranscription. Those DNA sequences are referred to herein astranscription-termination regions. Those regions are required forefficient polyadenylation of transcribed messenger RNA (mRNA). Examplesof preferred 3′ regions are (1) the 3′ transcribed, non-translatedregions containing the polyadenylation signal of Agrobacteriumtumor-inducing (Ti) plasmid genes, such as the nopaline synthase (NOS)gene and (2) the 3′ ends of plant genes such as the pearibulose-1,5-bisphosphate carboxylase small subunit gene, designatedherein as E9 (Fischhoff et al., 1987). Constructs will typically includethe gene of interest along with a 3′ end DNA sequence that acts as asignal to terminate transcription and, in constructs intended fornuclear genome expression, allow for the poly-adenylation of theresultant mRNA. The most preferred 3′ elements are contemplated to bethose from the nopaline synthase gene of A. tumefaciens (nos 3′end)(Bevan et al., 1983), the terminator for the T7 transcript from theoctopine synthase gene of A. tumefaciens, and the 3′ end of the proteaseinhibitor I or II genes from potato or tomato. Regulatory elements suchas TMV Ω element (Gallie, et al., 1989), may further be included wheredesired.

[0114] According to the present invention and as noted above,chloroplast or plastid localized genes encoding enzymes conferringherbicide tolerance or herbicide resistance characteristics to plants donot require sequences which confer transcription termination andpolyadenylation signals, but instead may only require transcriptiontermination information at the 3′ end of the gene. For coding sequencesintroduced into a chloroplast or plastid, or into a chloroplast orplastid genome, mRNA transcription termination is similar to methodswell known in the bacterial gene expression art. For example, either ina polycistronic or a monocistronic sequence, transcription can beterminated by stem and loop structures or by structures similar to rhodependent sequences.

[0115] Transcription enhancers or duplications of enhancers could beused to increase expression. These enhancers often are found 5′ to thestart of transcription in a promoter that functions in eukaryotic cells,but can often be inserted in the forward or reverse orientation 5′ or 3′to the coding sequence. Examples of enhancers include elements from theCaMV 35S promoter, octopine synthase genes (Ellis et al., 1987), therice actin gene, and promoter from non-plant eukaryotes (e.g., yeast; Maet al., 1988).

[0116] In certain embodiments of the invention, the use of internalribosome binding sites (IRES) elements are used to create multigene, orpolycistronic, messages. IRES elements are able to bypass the ribosomescanning model of 5′ methylated Cap dependent translation and begintranslation at internal sites (Pelletier and Sonenberg, 1988). IRESelements from two members of the picomavirus family (polio andencephalomyocarditis) have been described (Pelletier and Sonenberg,1988), as well an IRES from a mammalian message (Macejak and Sarnow,1991). IRES elements can be linked to heterologous open reading frames.Multiple open reading frames can be transcribed together, each separatedby an IRES, creating polycistronic messages. By virtue of the IRESelement, each open reading frame is accessible to ribosomes forefficient translation. Multiple genes can be efficiently expressed usinga single promoter/enhancer to transcribe a single message.

[0117] Any heterologous open reading frame can be linked to IRESelements. This includes genes for secreted proteins, multi-subunitproteins, encoded by independent genes, intracellular or membrane-boundproteins and selectable markers. In this way, expression of severalproteins can be simultaneously engineered into a cell with a singleconstruct and a single selectable marker.

[0118] Constructs intended for expression from within a chloroplast orplastid utilizing chloroplast or plastid specific transcriptional andtranslational machinery can contain either mono- or polycistronicsequences.

[0119] The choice of which expression vector and ultimately to whichpromoter a polypeptide coding region is operatively linked dependsdirectly on the functional properties desired, e.g., the location andtiming of protein expression, and the host cell to be transformed. Theseare well known limitations inherent in the art of constructingrecombinant DNA molecules. However, a vector useful in practicing thepresent invention is capable of directing the expression of thepolypeptide coding region to which it is operatively linked.

[0120] Typical vectors useful for expression of genes in higher plantsare well known in the art and include vectors derived from thetumor-inducing (Ti) plasmid of A. tumefaciens described (Rogers et al.,1987). However, several other plant integrating vector systems are knownto function in plants including pCaMVCN transfer control vectordescribed (Fromm et al., 1985). pCaMVCN (available from Pharmacia,Piscataway, N.J.) includes the CaMV35S promoter.

[0121] In preferred embodiments, the vector used to express thepolypeptide includes a selection marker that is effective in a plantcell, preferably a drug resistance selection marker. One preferred drugresistance marker is the gene whose expression results in kanamycinresistance; i.e. the chimeric gene containing the nopaline synthasepromoter, Tn5 neomycin phosphotransferase II (nptII) and nopalinesynthase 3′ non-translated region described (Rogers et al., 1988).

[0122] Means for preparing expression vectors are well known in the art.Expression (transformation) vectors used to transform plants and methodsof making those vectors are described in U.S. Pat. Nos. 4,971,908,4,940,835, 4,769,061 and 4,757,011 (each of which is specificallyincorporated herein by reference). Those vectors can be modified toinclude a coding sequence in accordance with the present invention.

[0123] A variety of methods have been developed to operatively link DNAto vectors via complementary cohesive termini or blunt ends. Forinstance, complementary homopolymer tracts can be added to the DNAsegment to be inserted and to the vector DNA. The vector and DNA segmentare then joined by hydrogen bonding between the complementaryhomopolymeric tails to form recombinant DNA molecules.

[0124] A coding region that encodes a polypeptide having the ability toconfer enhanced herbicide resistance enzymatic activity to a cell ispreferably a polynucleotide encoding an AMPA transacylase or afunctional equivalent, alone or in combination, with a gene encoding aGOX enzyme or a functional equivalent of GOX. In accordance with suchembodiments, a coding region comprising the DNA sequence of SEQ ID NO:3, SEQ ID NO: 7, or SEQ ID NO: 19 is also preferred.

[0125] Specific genes encoding AMPA transacylase that have been shown tosuccessfully transform plants in conjunction with plastid targetingpeptide-encoding genes, to express the AMPA transacylase at sufficientherbicidally protective levels are those genes comprised within theplasmid vectors. Preferred plasmids containing plastid targetingsequences include pMON17261, pMON10151, pMON10149, pMON32570, pMON32571,pMON32572, pMON32573, pMON32926, pMON32931, pMON32932, pMON32936,pMON32938, pMON32946, pMON32947, pMON32948, and pMON32950. Theseplasmids contain polynucleotide sequences which encode targetingsequences as shown in SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ IDNO: 14. Expression cassettes comprising plant operable promoters linkedto coding sequences, some with and some without 5′ untranslatedsequences and/or intron sequences, wherein the coding sequences containat least an AMPA transacylase or transacetylase, linked to plantoperable termination sequences are disclosed in particular as set forthin SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, and SEQID NO: 31.

[0126] The work described herein has identified methods of potentiatingin planta expression of an AMPA transacylase, which confer protectionfrom glyphosate and related herbicides to plants when incorporated intothe nuclear, plastid, or chloroplast genome of susceptible plants whichalso express a GOX or similar gene. U.S. Pat. No. 5,500,365(specifically incorporated herein by reference) describes a method forsynthesizing plant genes to optimize the expression level of the proteinfor which the synthesized gene encodes. This method relates to themodification of the structural gene sequences of the exogenoustransgene, to make them more “plant-like” and therefore more likely tobe translated and expressed by the plant. A similar method for enhancedexpression of transgenes, preferably in monocotyledonous plants, isdisclosed in U.S. Pat. No. 5,689,052 (specifically incorporated hereinby reference). Agronomic, horticultural, ornamental, and othereconomically or commercially useful plants can be made in accordancewith the methods described herein.

[0127] Such plants may co-express the AMPA transacylase gene and/or aGOX gene along with other antifungal, antibacterial, or antiviralpathogenesis-related peptides, polypeptides, or proteins; insecticidalproteins; other proteins conferring herbicide resistance; and proteinsinvolved in improving the quality of plant products or agronomicperformance of plants. Simultaneous co-expression of multipleheterologous proteins in plants is advantageous in that it can exploitsmore than one mode of action to control plant damage or improve thequality of the plant or products produced by the plants metabolism.

[0128] It is contemplated that introduction of large DNA sequencescomprising more than one gene may be desirable. Introduction of suchsequences may be facilitated by use of bacterial or yeast artificialchromosomes (BACs or YACs, respectively), or even plant artificialchromosomes. For example, the use of BACs for Agrobacterium-mediatedtransformation was disclosed by Hamilton et al. (1996).

[0129] Ultimately, the most desirable DNA sequences for introductioninto a monocot genome may be homologous genes or gene families whichencode a desired trait (for example, increased yield), and which areintroduced under the control of novel promoters or enhancers, etc., orperhaps even homologous or tissue specific (e.g., root-collar/sheath-,whorl-, stalk-, earshank-, kernel- or leaf-specific) promoters orcontrol elements. Indeed, it is envisioned that a particular use of thepresent invention may be the production of transformants comprising atransgene which is targeted in a tissue-specific manner. For example,herbicide resistance or herbicide tolerance genes may be expressedspecifically or specifically regulated in a negative manner in theplants reproductive tissues which can provide a means for enhancingherbicide tolerance or sensitivity to those tissues. Such regulatorycontrol means can provide methods for regulating the escape oftransgenes into the environment or for controlling the illicit use ofproprietary or licensed intellectual or commercialized property.

[0130] Vectors for use in tissue-specific targeting of gene expressionin transgenic plants typically will include tissue-specific promotersand also may include other tissue-specific control elements such asenhancer sequences. Promoters which direct specific or enhancedexpression in certain plant tissues will be known to those of skill inthe art in light of the present disclosure.

[0131] It also is contemplated that tissue specific expression may befunctionally accomplished by introducing a constitutively expressed gene(all tissues) in combination with an antisense gene that is expressedonly in those tissues where the gene product is not desired. Forexample, a gene coding for the AMPA transacylase from E. coli may beintroduced such that it is expressed in all tissues using the 35Spromoter from Cauliflower Mosaic Virus. Alternatively, a rice actinpromoter or a histone promoter from a dicot or monocot species alsocould be used for constitutive expression of a gene. Furthermore, it iscontemplated that promoters combining elements from more than onepromoter may be useful. For example, U.S. Pat. No. 5,491,288 disclosescombining a Cauliflower Mosaic Virus promoter with a histone promoter.Therefore, expression of an antisense transcript of the AMPAtransacylase gene in a maize kernel, using for example a zein promoter,would prevent accumulation of the transacylase in seed. Thus, in a plantexpressing both GOX and the transacylase, application of glyphosateherbicide would result in seed tissues which fail to mature. Conversely,antisense suppression of the GOX gene would effectuate the same result.Preferably, suppression of the transacylase in specific tissues would bemore advantageous, particularly where specific tissues have demonstratedan intolerance to AMPA or related compounds. It is specificallycontemplated by the inventor that a similar strategy could be used withthe instant invention to direct expression of a screenable or selectablemarker in seed tissue.

[0132] Alternatively, one may wish to obtain novel tissue-specificpromoter sequences for use in accordance with the present invention. Toachieve this, one may first isolate cDNA clones from the tissueconcerned and identify those clones which are expressed specifically inthat tissue, for example, using Northern blotting. Ideally, one wouldlike to identify a gene that is not present in a high copy number, butwhich gene product is relatively abundant in specific tissues. Thepromoter and control elements of corresponding genomic clones may thisbe localized using the techniques of molecular biology known to those ofskill in the art.

[0133] It is contemplated that expression of some genes in transgenicplants will be desired only under specified conditions. For example, itis proposed that expression of certain genes that confer resistance toenvironmentally stress factors such as drought will be desired onlyunder actual stress conditions. It further is contemplated thatexpression of such genes throughout a plants development may havedetrimental effects. It is known that a large number of genes exist thatrespond to the environment. For example, expression of some genes suchas rbcS, encoding the small subunit of ribulose bisphosphatecarboxylase, is regulated by light as mediated through phytochrome.Other genes are induced by secondary stimuli. For example, synthesis ofabscisic acid (ABA) is induced by certain environmental factors,including but not limited to water stress. A number of genes have beenshown to be induced by ABA (Skriver and Mundy, 1990). It also isexpected that expression of genes conferring resistance to applicationsof herbicides would be desired only under conditions in which herbicideis actually present. Therefore, for some desired traits, inducibleexpression of genes in transgenic plants will be desired.

[0134] It is proposed that, in some embodiments of the presentinvention, expression of a gene in a transgenic plant will be desiredonly in a certain time period during the development of the plant.Developmental timing frequently is correlated with tissue specific geneexpression. For example expression of zein storage proteins is initiatedin the endosperm about 15 days after pollination.

[0135] It also is contemplated that it may be useful to specificallytarget DNA insertion within a cell. For example, it may be useful totarget introduced DNA to the nucleus, and in particular into a preciseposition within one of the plant chromosomes in order to achieve sitespecific integration. For example, it would be useful to have a geneintroduced through transformation which acts to replace an existing genein the cell, or to complement a gene which is not functional or presentat all.

[0136] A plant transformed with an expression vector of the presentinvention is also contemplated. A transgenic plant derived from such atransformed or transgenic cell is also contemplated. Those skilled inthe art will recognize that a chimeric plant gene containing astructural coding sequence of the present invention can be inserted intothe genome of a plant by methods well known in the art. Such methods forDNA transformation of plant cells include Agrobacterium-mediated planttransformation, the use of liposomes, transformation using viruses orpollen, electroporation, protoplast transformation, gene transfer intopollen, injection into reproductive organs, injection into immatureembryos and particle bombardment. Each of these methods has distinctadvantages and disadvantages. Thus, one particular method of introducinggenes into a particular plant strain may not necessarily be the mosteffective for another plant strain, but it is well known which methodsare useful for a particular plant strain.

[0137] There are many methods for introducing transforming DNA segmentsinto cells, but not all are suitable for delivering DNA to plant cells.Suitable methods are believed to include virtually any method by whichDNA can be introduced into a cell, such as infection by A. tumefaciensand related Agrobacterium strains, direct delivery of DNA such as, forexample, by PEG-mediated transformation of protoplasts (Omirulleh etal., 1993), by desiccation/inhibition-mediated DNA uptake, byelectroporation, by agitation with silicon carbide fibers, byacceleration of DNA coated particles, etc. In certain embodiments,acceleration methods are preferred and include, for example,microprojectile bombardment and the like.

[0138] Technology for introduction of DNA into cells is well-known tothose of skill in the art. Four general methods for delivering a geneinto cells have been described: (1) chemical methods (Graham and van derEb, 1973); (2) physical methods such as microinjection (Capecchi, 1980),electroporation (Wong and Neumann, 1982; Fromm et al., 1985) and thegene gun (Johnston and Tang, 1994; Fynan et al., 1993); (3) viralvectors (Clapp, 1993; Lu et al., 1993; Eglitis and Anderson, 1988a;1988b); and (4) receptor-mediated mechanisms (Curiel et al., 1991; 1992;Wagner et al., 1992).

[0139] The application of brief, high-voltage electric pulses to avariety of animal and plant cells leads to the formation ofnanometer-sized pores in the plasma membrane. DNA is taken directly intothe cell cytoplasm either through these pores or as a consequence of theredistribution of membrane components that accompanies closure of thepores. Electroporation can be extremely efficient and can be used bothfor transient expression of cloned genes and for establishment of celllines that carry integrated copies of the gene of interest.Electroporation, in contrast to calcium phosphate-mediated transfectionand protoplast fusion, frequently gives rise to cell lines that carryone, or at most a few, integrated copies of the foreign DNA.

[0140] The introduction of DNA by means of electroporation is well-knownto those of skill in the art. To effect transformation byelectroporation, one may employ either friable tissues such as asuspension culture of cells, or embryogenic callus, or alternatively,one may transform immature embryos or other organized tissues directly.One would partially degrade the cell walls of the chosen cells byexposing them to pectin-degrading enzymes (pectolyases) or mechanicallywounding in a controlled manner, rendering the cells more susceptible totransformation. Such cells would then be recipient to DNA transfer byelectroporation, which may be carried out at this stage, and transformedcells then identified by a suitable selection or screening protocoldependent on the nature of the newly incorporated DNA.

[0141] A further advantageous method for delivering transforming DNAsegments to plant cells is microprojectile bombardment. In this method,particles may be coated with nucleic acids and delivered into cells by apropelling force. Exemplary particles include those comprised oftungsten, gold, platinum, and the like. Using these particles, DNA iscarried through the cell wall and into the cytoplasm on the surface ofsmall metal particles as described (Klein et al., 1987; Klein et al.,1988; Kawata et al., 1988). The metal particles penetrate throughseveral layers of cells and thus allow the transformation of cellswithin tissue explants. The microprojectile bombardment method ispreferred for the identification of chloroplast or plastid directedtransformation events.

[0142] An advantage of microprojectile bombardment, in addition to itbeing an effective means of reproducibly stably transforming plantcells, is that neither the isolation of protoplasts (Cristou et al.,1988) nor the susceptibility to Agrobacterium infection is required. Anillustrative embodiment of a method for delivering DNA into plant cellsby acceleration is a Biolistics Particle Delivery System, which can beused to propel particles coated with DNA or cells through a screen, suchas a stainless steel or Nytex screen, onto a filter surface covered withthe plant cultured cells in suspension. The screen disperses theparticles so that they are not delivered to the recipient cells in largeaggregates. It is believed that a screen intervening between theprojectile apparatus and the cells to be bombarded reduces the size ofprojectiles aggregate and may contribute to a higher frequency oftransformation by reducing damage inflicted on the recipient cells byprojectiles that are too large.

[0143] For the bombardment, cells in suspension are preferablyconcentrated on filters or solid culture medium. Alternatively, immatureembryos or other target cells may be arranged on solid culture medium.The cells to be bombarded are positioned at an appropriate distancebelow the microprojectile stopping plate. If desired, one or morescreens are also positioned between the acceleration device and thecells to be bombarded. Through the use of techniques set forth hereinone may obtain up to 1000 or more foci of cells transiently expressing amarker gene. The number of cells in a focus which express the exogenousgene product 48 hours post-bombardment often range from 1 to 10 andaverage 1 to 3.

[0144] In bombardment transformation, one may optimize thepre-bombardment culturing conditions and the bombardment parameters toyield the maximum numbers of stable transformants. Both the physical andbiological parameters for bombardment are important in this technology.Physical factors are those that involve manipulating theDNA/microprojectile precipitate or those that affect the flight andvelocity of either the macro- or microprojectiles. Biological factorsinclude all steps involved in manipulation of cells before andimmediately after bombardment, the osmotic adjustment of target cells tohelp alleviate the trauma associated with bombardment, and also thenature of the transforming DNA, such as linearized DNA or intactsupercoiled plasmids. It is believed that pre-bombardment manipulationsare especially important for successful transformation of immature plantembryos.

[0145] Accordingly, it is contemplated that one may desire to adjustvarious of the bombardment parameters in small scale studies to fullyoptimize the conditions. One may particularly wish to adjust physicalparameters such as gap distance, flight distance, tissue distance, andhelium pressure. One may also minimize the trauma reduction factors(TRFs) by modifying conditions which influence the physiological stateof the recipient cells and which may therefore influence transformationand integration efficiencies. For example, the osmotic state, tissuehydration and the subculture stage or cell cycle of the recipient cellsmay be adjusted for optimum transformation. The execution of otherroutine adjustments will be known to those of skill in the art in lightof the present disclosure.

[0146] The methods of particle-mediated transformation is well-known tothose of skill in the art. U.S. Pat. No. 5,015,580 (specificallyincorporated herein by reference) describes the transformation ofsoybeans using such a technique.

[0147] Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells because the DNA can be introducedinto whole plant tissues, thereby bypassing the need for regeneration ofan intact plant from a protoplast. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art. See, for example, the methods described (Fraley etal., 1985; Rogers et al, 1987). The genetic engineering of cotton plantsusing Agrobacterium-mediated transfer is described in U.S. Pat. No.5,004,863 (specifically incorporated herein by reference); liketransformation of lettuce plants is described in U.S. Pat. No. 5,349,124(specifically incorporated herein by reference); and theAgrobacterium-mediated transformation of soybean is described in U.S.Pat. No. 5,416,011 (specifically incorporated herein by reference).Further, the integration of the Ti-DNA is a relatively precise processresulting in few rearrangements. The region of DNA to be transferred isdefined by the border sequences, and intervening DNA is usually insertedinto the plant genome as described (Spielmann et al., 1986; Jorgensen etal., 1987).

[0148] Modern Agrobacterium transformation vectors are capable ofreplication in E. coli as well as Agrobacterium, allowing for convenientmanipulations as described (Klee et al., 1985). Moreover, recenttechnological advances in vectors for Agrobacterium-mediated genetransfer have improved the arrangement of genes and restriction sites inthe vectors to facilitate construction of vectors capable of expressingvarious polypeptide coding genes. The vectors described (Rogers et al.,1987), have convenient multi-linker regions flanked by a promoter and apolyadenylation site for direct expression of inserted polypeptidecoding genes and are suitable for present purposes. In addition,Agrobacterium containing both armed and disarmed Ti genes can be usedfor the transformations. In those plant varieties whereAgrobacterium-mediated transformation is efficient, it is the method ofchoice because of the facile and defined nature of the gene transfer.

[0149] Agrobacterium-mediated transformation of leaf disks and othertissues such as cotyledons and hypocotyls appears to be limited toplants that Agrobacterium naturally infects. Agrobacterium-mediatedtransformation is most efficient in dicotyledonous plants. Few monocotsappear to be natural hosts for Agrobacterium, although transgenic plantshave been produced in asparagus using Agrobacterium vectors as described(Bytebier et al., 1987). Other monocots recently have also beentransformed with Agrobacterium. Included in this group are corn (Ishidaet al.) and rice (Cheng et al.).

[0150] A transgenic plant formed using Agrobacterium transformationmethods typically contains a single gene on one chromosome. Suchtransgenic plants can be referred to as being heterozygous for the addedgene. However, inasmuch as use of the word “heterozygous” usuallyimplies the presence of a complementary gene at the same locus of thesecond chromosome of a pair of chromosomes, and there is no such gene ina plant containing one added gene as here, it is believed that a moreaccurate name for such a plant is an independent segregant, because theadded. exogenous gene segregates independently during mitosis andmeiosis.

[0151] An independent segregant may be preferred when the plant iscommercialized as a hybrid, such as corn. In this case, an independentsegregant containing the gene is crossed with another plant, to form ahybrid plant that is heterozygous for the gene of interest.

[0152] An alternate preference is for a transgenic plant that ishomozygous for the added structural gene; i.e. a transgenic plant thatcontains two added genes, one gene at the same locus on each chromosomeof a chromosome pair. A homozygous transgenic plant can be obtained bysexually mating (selfing) an independent segregant transgenic plant thatcontains a single added gene, germinating some of the seed produced andanalyzing the resulting plants produced for gene of interest activityand mendelian inheritance indicating homozygosity relative to a control(native, non-transgenic) or an independent segregant transgenic plant.

[0153] Two different transgenic plants can be mated to produce offspringthat contain two independently segregating added, exogenous genes.Selfing of appropriate progeny can produce plants that are homozygousfor both added, exogenous genes that encode a polypeptide of interest.Back-crossing to a parental plant and out-crossing with a non-transgenicplant are also contemplated.

[0154] Transformation of plant protoplasts can be achieved using methodsbased on calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments (see e.g.,Potrykus et al, 1985; Lorz et al., 1985; Fromm et al., 1985; Uchimiya etal., 1986; Callis et al., 1987; Marcotte et al., 1988).

[0155] Application of these systems to different plant gernplasm dependsupon the ability to regenerate that particular plant variety fromprotoplasts. Illustrative methods for the regeneration of cereals fromprotoplasts are described (see, e.g., Fujimura et al., 1985; Toriyama etal., 1986; Yamada et al., 1986; Abdullah et al., 1986).

[0156] To transform plant germplasm that cannot be successfullyregenerated from protoplasts, other ways to introduce DNA into intactcells or tissues can be utilized. For example, regeneration of cerealsfrom immature embryos or explants can be effected as described (Vasil,1988).

[0157] Unmodified bacterial genes are often poorly expressed intransgenic plant cells. Plant codon usage more closely resembles that ofhumans and other higher organisms than unicellular organisms, such asbacteria. Several reports have disclosed methods for improvingexpression of recombinant genes in plants (Murray et al., 1989; Diehn etal., 1996; Iannacone et al., 1997; Rouwendal et al., 1997; Futterer etal., 1997; and Futterer and Hohn, 1996). These reports disclose variousmethods for engineering coding sequences to represent sequences whichare more efficiently translated based on plant codon frequency tables,improvements in codon third base position bias, using recombinantsequences which avoid suspect polyadenylation or A/T rich domains orintron splicing consensus sequences.

[0158] U.S. Pat. No. 5,500,365 (specifically incorporated herein byreference) describes the preferred method for synthesizing plant genesto optimize the expression level of the protein for which thesynthesized gene encodes. This method relates to the modification of thestructural gene sequences of the exogenous transgene, to make them more“plant-like” and therefore more likely to be translated and expressed bythe plant, monocot or dicot. However, the method as disclosed in U.S.Pat. No. 5,689,052 provides for enhanced expression of transgenes,preferably in monocotyledonous plants, which is herein incorporated inits entirety by reference. Briefly, according to Brown et al., thefrequency of rare and semi-rare monocotyledonous codons in apolynucleotide sequence encoding a desired protein are reduced andreplaced with more preferred monocotyledonous codons. Enhancedaccumulation of a desired polypeptide encoded by a modifiedpolynucleotide sequence in a monocotyledonous plant is the result ofincreasing the frequency of preferred codons by analyzing the codingsequence in successive six nucleotide fragments and altering thesequence based on the frequency of appearance of the six-mers as to thefrequency of appearance of the rarest 284, 484, and 664 six-mers inmonocotyledenous plants. Furthermore, Brown et al. disclose the enhancedexpression of a recombinant gene by applying the method for reducing thefrequency of rare codons with methods for reducing the occurrence ofpolyadenylation signals and intron splice sites in the nucleotidesequence, removing self-complementary sequences in the nucleotidesequence and replacing such sequences with nonself-complementarynucleotides while maintaining a structural gene encoding thepolypeptide, and reducing the frequency of occurrence of 5′-CG-3′di-nucleotide pairs in the nucleotide sequence. These steps areperformed sequentially and have a cumulative effect resulting in anucleotide sequence containing a preferential utilization of themore-preferred monocotyledonous codons for monocotyledonous plants for amajority of the amino acids present in the desired polypeptide.

[0159] Thus, the amount of a gene coding for a polypeptide of interestcan be increased in plants by transforming those plants usingtransformation methods such as those disclosed herein. In particular,chloroplast or plastid transformation can result in desired codingsequences being present in up to about 10,000 copies per cell in tissuescontaining these subcellular organelle structures (McBride et al.,Bio/Technology 13:362-365, 1995).

[0160] DNA can also be introduced into plants by direct DNA transferinto pollen as described (Zhou et al., 1983; Hess, 1987). Expression ofpolypeptide coding genes can be obtained by injection of the DNA intoreproductive organs of a plant as described (Pena et al., 1987). DNA canalso be injected directly into the cells of immature embryos andintroduced into cells by rehydration of desiccated embryos as described(Neuhaus et al., 1987; Benbrook et al., 1986).

[0161] After effecting delivery of exogenous DNA to recipient cells, thenext step to obtain a transgenic plant generally concern identifying thetransformed cells for further culturing and plant regeneration. Asmentioned herein, in order to improve the ability to identifytransformants, one may desire to employ a selectable or screenablemarker gene as, or in addition to, the expressible gene of interest. Inthis case, one would then generally assay the potentially transformedcell population by exposing the cells to a selective agent or agents, orone would screen the cells for the desired marker gene trait.

[0162] An exemplary embodiment of methods for identifying transformedcells involves exposing the transformed cultures to a selective agent,such as a metabolic inhibitor, an antibiotic, herbicide or the like.Cells which have been transformed and have stably integrated a markergene conferring resistance to the selective agent used, will grow anddivide in culture. Sensitive cells will not be amenable to furtherculturing. One example of a preferred marker gene confers resistance toglyphosate. When this gene is used as a selectable marker, theputatively transformed cell culture is treated with glyphosate. Upontreatment, transgenic cells will be available for further culturingwhile sensitive, or non-transformed cells, will not. This method isdescribed in detail in U.S. Pat. No. 5,569,834, which is specificallyincorporated herein by reference. Another example of a preferredselectable marker system is the neomycin phosphotransferase (nptII)resistance system by which resistance to the antibiotic kanamycin isconferred, as described in U.S. Pat. No. 5,569,834 (specificallyincorporated herein by reference). Again, after transformation with thissystem, transformed cells will be available for further culturing upontreatment with kanamycin, while non-transformed cells will not. Yetanother preferred selectable marker system involves the use of a geneconstruct conferring resistance to paromomycin. Use of this type of aselectable marker system is described in U.S. Pat. No. 5,424,412(specifically incorporated herein by reference).

[0163] Another preferred selectable marker system involves the use ofthe genes contemplated by this invention. In particular, a phnO gene ora substantially similar gene encoding an AMPA transacylase can beutilized as a selectable marker. Plant cells which have had arecombinant DNA molecule introduced into their genome can be selectedfrom a population of cells which failed to incorporate a recombinantmolecule by growing the cells in the presence of AMPA. One skilled inthe art will recognize the particular advantages that this selectablemarker system has over previous selectable marker systems. Theselectable marker used in the recombinant DNA integrated into a plantgenome reduces the amount of DNA targeted for integration because theselectable marker will also be used for improved herbicide tolerance orimproved herbicide resistance in plants generated from transformed plantcells. This selectable marker also provides an additional marker systemnot known before, particularly in a field in which there are often onlya limited number of selectable markers available.

[0164] Transplastonomic selection (selection of plastid or chloroplasttransformation events) is simplified by taking advantage of thesensitivity of chloroplasts or plastids to spectinomycin, an inhibitorof plastid or chloroplast protein synthesis, but not of proteinsynthesis by the nuclear genome encoded cytoplasmic ribosomes.Spectinomycin prevents the accumulation of chloroplast proteins requiredfor photosynthesis and so spectinomycin resistant transformed plantcells may be distinguished on the basis of their difference in color:the resistant, transformed cells are green, whereas the sensitive cellsare white, due to inhibition of plastid-protein synthesis.Transformation of chloroplasts or plastids with a suitable bacterial aadgene, or with a gene encoding a spectinomycin resistant plastid orchloroplast functional ribosomal RNA provides a means for selection andmaintenance of transplastonomic events (Maliga, Trends in Biotechnology11:101-106, 1993).

[0165] It is further contemplated that combinations of screenable andselectable markers will be useful for identification of transformedcells. In some cell or tissue types a selection agent, such asglyphosate or kanamycin, may either not provide enough killing activityto clearly recognize transformed cells or may cause substantialnonselective inhibition of transformants and nontransformants alike,thus causing the selection technique to not be effective. It is proposedthat selection with a growth inhibiting compound, such as glyphosate atconcentrations below those that cause 100% inhibition followed byscreening of growing tissue for expression of a screenable marker genesuch as kanamycin would allow one to recover transformants from cell ortissue types that are not amenable to selection alone. It is proposedthat combinations of selection and screening may enable one to identifytransformants in a wider variety of cell and tissue types. Theavailability of the transacylases of the present invention may obviatethe necessity for combination selection and screening by providing anadditional selection means.

[0166] The development or regeneration of plants from either singleplant protoplasts or various explants is well known in the art(Weissbach and Weissbach, 1988). This regeneration and growth processtypically includes the steps of selection of transformed cells,culturing those individualized cells through the usual stages ofembryonic development through the rooted plantlet stage. Transgenicembryos and seeds are similarly regenerated. The resulting transgenicrooted shoots are thereafter planted in an appropriate plant growthmedium such as soil.

[0167] The development or regeneration of plants containing the foreign,exogenous gene that encodes a polypeptide of interest introduced byAgrobacterium from leaf explants can be achieved by methods well knownin the art such as described (Horsch et al., 1985). In this procedure,transformants are cultured in the presence of a selection agent and in amedium that induces the regeneration of shoots in the plant strain beingtransformed as described (Fraley et al., 1983). In particular, U.S. Pat.No. 5,349,124 (specification incorporated herein by reference) detailsthe creation of genetically transformed lettuce cells and plantsresulting therefrom which express hybrid crystal proteins conferringinsecticidal activity against Lepidopteran larvae to such plants.

[0168] This procedure typically produces shoots within two to fourmonths and those shoots are then transferred to an appropriateroot-inducing medium containing the selective agent and an antibiotic toprevent bacterial growth. Shoots that rooted in the presence of theselective agent to form plantlets are then transplanted to soil or othermedia to allow the production of roots. These procedures vary dependingupon the particular plant strain employed, such variations being wellknown in the art.

[0169] Preferably, the regenerated plants are self-pollinated to providehomozygous transgenic plants, or pollen obtained from the regeneratedplants is crossed to seed-grown plants of agronomically important,preferably inbred lines. Conversely, pollen from plants of thoseimportant lines is used to pollinate regenerated plants. A transgenicplant of the present invention containing a desired polypeptide iscultivated using methods well known to one skilled in the art.

[0170] In one embodiment, a transgenic plant of this invention thus hasan increased amount of a coding region encoding an AMPA transacylasepolypeptide which may also be expressed along with a plastid targetingpeptide. A preferred transgenic plant is an independent segregant andcan transmit that gene and its activity to its progeny. A more preferredtransgenic plant is homozygous for that gene, and transmits that gene toall of its offspring on sexual mating. Seed from a transgenic plant maybe grown in the field or greenhouse, and resulting sexually maturetransgenic plants are self-pollinated to generate true breeding plants.The progeny from these plants become true breeding lines that areevaluated for expression of the transacylase transgene as well as forimproved herbicide tolerance, particularly when the transacylasetransgene is co-expressed along with a gene encoding a GOX enzyme.

[0171] The genes and acyltransferases according to the subject inventioninclude not only the full length sequences disclosed herein but alsofragments of these sequences, or fusion proteins, which retain thecharacteristic improved herbicidal protective activity of the sequencesspecifically exemplified herein.

[0172] It should be apparent to a person of skill in this art that AMPAtransacylase genes and peptides can be identified and obtained throughseveral means. The specific genes, or portions thereof, may be obtainedfrom a culture depository, or constructed synthetically, for example, byuse of a gene machine. Variations of these genes may be readilyconstructed using standard techniques for making point mutations. Also,fragments of these genes can be made using commercially availableexonucleases or endonucleases according to standard procedures. Forexample, enzymes such as Bal31 or site-directed mutagenesis can be usedto systematically cut off nucleotides from the ends of these genes.Also, genes which code for active fragments may be obtained using avariety of other restriction enzymes. Proteases may be used to directlyobtain active fragments of such transacylases.

[0173] Equivalent AMPA transacylases and/or genes encoding thesetransacylases can also be isolated from E. coli strains and/or DNAlibraries using the teachings provided herein. For example, antibodiesto the transacylases disclosed and claimed herein can be used toidentify and isolate other transacylases from a mixture of proteins.Specifically, antibodies may be raised to the transacylases disclosedherein and used to specifically identify equivalent AMPA transacylasesby immunoprecipitation, column immuno-purification, enzyme linkedimmunoassay (ELISA), or Western blotting.

[0174] A further method for identifying the peptides and genes of thesubject invention is through the use of oligonucleotide probes. Theseprobes are nucleotide sequences having a detectable label. As is wellknown in the art, if the probe molecule and sequences in a targetnucleic acid sample hybridize by forming a strong bond between the twomolecules, it can be reasonably assumed that the probe and target samplecontain essentially identical polynucleotide sequences. The probe'sdetectable label provides a means for determining in a known mannerwhether hybridization has occurred. Such a probe analysis provides arapid method for identifying AMPA transacylase genes of the subjectinvention.

[0175] The nucleotide segments which are used as probes according to theinvention can be synthesized by use of DNA synthesizers using standardprocedures. In the use of the nucleotide segments as probes, theparticular probe is labeled with any suitable label known to thoseskilled in the art, including radioactive and non-radioactive labels.Typical radioactive labels include ³²P, ¹²⁵I, ³⁵S, or the like. A probelabeled with a radioactive isotope can be constructed from a nucleotidesequence complementary to the DNA sample by a conventional nicktranslation reaction, using a DNase and DNA polymerase. The probe andsample can then be combined in a hybridization buffer solution and heldat an appropriate temperature until annealing occurs. Thereafter, themembrane is washed free of extraneous materials, leaving the sample andbound probe molecules typically detected and quantified byautoradiography and/or liquid scintillation counting.

[0176] Non-radioactive labels include, for example, ligands such asbiotin or thyroxin, as well as enzymes such as hydrolyses orperoxidases, or the various chemiluminescers such as luciferin, orfluorescent compounds like fluorescein, rhodamine, Texas Red, andderivatives and the like. The probe may also be labeled at both endswith different types of labels for ease of separation, as, for example,by using an isotopic label at the end mentioned above and a biotin labelat the other end, or with different fluorescent emitters which haveoverlapping absorption and emission spectra.

[0177] Duplex formation and stability depend on substantialcomplementary between the two strands of a hybrid, and, as noted above,a certain degree of mismatch can be tolerated. Therefore, the probes ofthe subject invention include mutations (both single and multiple),deletions, insertions of the described sequences, and combinationsthereof, wherein said mutations, insertions and deletions permitformation of stable hybrids with the target polynucleotide of interest.Mutations, insertions, and deletions can be produced in a givenpolynucleotide sequence in many ways, by methods currently known to anordinarily skilled artisan, and perhaps by other methods which maybecome known in the future.

[0178] The potential variations in the probes listed is due, in part, tothe redundancy of the genetic code. Because of the redundancy of thegenetic code, more than one coding nucleotide triplet (codon) can beused for most of the amino acids used to make proteins. Thereforedifferent nucleotide sequences can code for a particular amino acid.Thus, the amino acid sequence of the E. coli AMPA transacylase andpeptide, and the plastid targeting peptides and the polynucleotideswhich code for them, can be prepared by equivalent nucleotide sequencesencoding the same amino acid sequence of the protein or peptide.Accordingly, the subject invention includes such equivalent nucleotidesequences. Also, inverse or complement sequences are an aspect of thesubject invention and can be readily used by a person skilled in thisart. In addition it has been shown that proteins of identified structureand function may be constructed by changing the amino acid sequence ifsuch changes do not alter the protein secondary structure (Kaiser andKezdy, 1984). Thus, the subject invention includes mutants of the aminoacid sequence depicted herein which do not alter the protein secondarystructure, or if the structure is altered, the biological activity issubstantially retained. Further, the invention also includes mutants oforganisms hosting all or part of a gene encoding an AMPA acyltransferaseand/or gene encoding a plastid targeting peptide, as discussed in thepresent invention. Such mutants can be made by techniques well known topersons skilled in the art. For example, UV irradiation can be used toprepare mutants of host organisms. Likewise, such mutants may includeasporogenous host cells which also can be prepared by procedures wellknown in the art.

[0179] Site-specific or site-directed mutagenesis is a technique usefulin the preparation of individual, novel and unique useful peptides, orbiologically functional equivalent proteins or peptides, throughspecific mutagenesis of structural genes encoding such peptides. Thetechnique further provides a ready ability to prepare and test sequencevariants by altering the coding sequence of a gene, for example, byintroducing one or more nucleotide sequence changes into the DNA for thepurpose of creating a new or useful restriction endonuclease cleavagerecognition sequence or for the purpose of altering the coding sequenceso that a gene's codons and percent G/C represent those more commonlyused by a particular genus or species. Site-specific mutagenesis allowsthe production of deletion, insertion, or replacement mutations throughthe use of specific mutagenesis oligonucleotide sequences comprising theDNA sequence of the desired mutation. Mutagenesis oligonucleotidestypically provide a primer sequence of sufficient size and sequencecomplexity to form a stable duplex on both sides of the desired mutationtarget site. Typically, a primer of about 17 to 25 nucleotides in lengthis preferred, with about 5 to 10 residues overlapping either side of thedesired mutation target site.

[0180] In general, the technique of site-specific mutagenesis is wellknown in the art, as exemplified by various publications. As will beappreciated, the technique typically employs a phage vector which existsin both a single stranded and double stranded form. Typical vectorsuseful in site-directed mutagenesis include vectors such as the M13phage. These phage are readily commercially available and their use isgenerally well known to those skilled in the art. Double strandedplasmids are also routinely employed in site directed mutagenesis, andoften contain a filamentous phage origin of replication which, in thepresence of a helper phage, allows synthesis of single stranded DNA fromthe plasmid vector.

[0181] In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector or melting apartof two strands of a double stranded vector which includes within itssequence a mutation target site. A mutagenesis oligonucleotide primerbearing the desired mutant sequence is prepared, generallysynthetically. The mutagenesis primer is then annealed with thesingle-stranded vector at the mutation target site, and subjected to DNApolymerizing enzymes such as E. coli polymerase I Klenow fragment, inorder to complete the synthesis of the mutation-bearing strand. Thus, aheteroduplex is formed wherein one strand encodes the originalnon-mutated sequence and the second strand bears the desired mutation.This heteroduplex vector is then used to transform appropriate cells,such as E. coli cells, and clones are selected which include recombinantvectors containing the mutation represented by the mutagenesis primersequence.

[0182] The preparation of sequence variants of the selectedpeptide-encoding DNA segments using site-directed mutagenesis isprovided as a means of producing potentially useful species and is notmeant to be limiting as there are other ways in which sequence variantsof peptides and the DNA sequences encoding them may be obtained. Forexample, recombinant vectors encoding the desired peptide sequence maybe treated with mutagenic agents, such as hydroxylamine, to obtainsequence variants. Such procedures may favorably change the protein'sbiochemical and biophysical characteristics or its mode of action. Theseinclude, but are not limited to: 1) improved AMPA transacylaseformation, 2) improved protein stability or reduced proteasedegradation, 3) improved substrate recognition and binding, 4) improvedenzyme kinetics, and 5) improved N-acyl-AMPA formation due to any or allof the reasons stated above.

[0183] Modification and changes may be made in the structure of thepeptides of the present invention and DNA segments which encode them andstill obtain a functional molecule that encodes a protein or peptidewith desirable characteristics. The biologically functional equivalentpeptides, polypeptides, and proteins contemplated herein should possessat least from about 40% to about 65% sequence similarity, preferablyfrom about 66% to about 75% sequence similarity, more preferably fromabout 76% to about 85% similarity, and most preferably from about 86% toabout 90% or greater sequence similarity to the sequence of, orcorresponding moiety within, the AMPA acyltransferase amino acidsequences disclosed herein.

[0184] The following is a discussion based upon changing the amino acidsof a protein to create an equivalent, or even an improved,second-generation molecule. In particular embodiments of the invention,mutated AMPA transacylase proteins are contemplated to be useful forimproving or enhancing the in planta expression of the protein, andconsequently increasing or improving the AMPA transacylase activityand/or expression of the recombinant transgene in a plant cell. Theamino acid changes may be achieved by changing the codons of the DNAsequence, according to the codons given in Table 1, in dicotyledonous,and more particularly in monocotyledonous plants. TABLE 1 Amino AcidCodons Alanine Ala A GCA GCC GCG GCU Cysteine Cys C UGC UGU Asparticacid Asp D GAC GAU Glutamic acid Glu E GAA GAG Phenylalanine Phe F UUCUUU Glycine Gly G GGA GGC GGG GGU Histidine His H CAC CAU Isoleucine IleI AUA AUC AUU Lysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUUMethionine Met M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCC CCGCCU Glutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGUSerine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACUValine Val V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

[0185] For example, certain amino acids may be substituted for otheramino acids in a protein structure without appreciable loss ofinteractive binding capacity with structures such as, for example,antigen-binding regions of antibodies or binding sites on substratemolecules. Since it is the interactive capacity and nature of a proteinthat defines that protein's biological functional activity, certainamino acid sequence substitutions can be made in a protein sequence,and, of course, its underlying DNA coding sequence, and neverthelessobtain a protein with like properties. It is thus contemplated by theinventor that various changes may be made in the peptide sequences ofthe disclosed compositions, or corresponding DNA sequences which encodesaid peptides without appreciable loss of their biological utility oractivity.

[0186] In making such changes, the hydropathic index of amino acids maybe considered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporate herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like.

[0187] Each amino acid has been assigned a hydropathic index on thebasis of their hydrophobicity and charge characteristics (Kyte andDoolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine(+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine(+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutarnate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine(−3.5); lysine (−3.9); and arginine (−4.5).

[0188] It is known in the art that certain amino acids may besubstituted by other amino acids having a similar hydropathic index orscore and still result in a protein with similar biological activity,i.e. still obtain a biological functionally equivalent protein. Inmaking such changes, the substitution of amino acids whose hydropathicindices are within ±2 is preferred, those which are within ±1 areparticularly preferred, and those within ±0.5 are even more particularlypreferred.

[0189] It is also understood in the art that the substitution of likeamino acids can be made effectively on the basis of hydrophilicity. U.S.Pat. No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein.

[0190] As detailed in U.S. Pat. No. 4,554,101, the followinghydrophilicity values have been assigned to amino acid residues:arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1);serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0);threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5);cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8);isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan(−3.4).

[0191] It is understood that an amino acid can be substituted foranother having a similar hydrophilicity value and still obtain abiologically equivalent, and in particular, an immunologicallyequivalent protein. In such changes, the substitution of amino acidswhose hydrophilicity values are within ±2 is preferred, those which arewithin ±1 are particularly preferred, and those within ±0.5 are evenmore particularly preferred.

[0192] As outlined above, amino acid substitutions are generallytherefore based on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions which take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

[0193] Polynucleotides encoding heterologous proteins are known by thoseskilled in the art, to often be poorly expressed when incorporated intothe nuclear DNA of transgenic plants (reviewed by Diehn et al., 1996).Preferably, a nucleotide sequence encoding a heterologous protein ofinterest is designed essentially as described in U.S. Pat. Nos.5,500,365 and 5,689,052 (each specifically incorporated herein byreference). Examples of nucleotide sequences useful for expressioninclude but are not limited to, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO:11, and SEQ ID NO: 19.

[0194] Substitutes for an amino acid within the fundamental polypeptidesequence can be selected from other members of the class to which thenaturally occurring amino acid belongs. Amino acids can be divided intothe following four groups: (1) acidic amino acids; (2) basic aminoacids; (3) neutral polar amino acids; and (4) neutral non-polar aminoacids. Representative amino acids within these various groups include,but are not limited to: (1) acidic (negatively charged) amino acids suchas aspartic acid and glutamic acid; (2) basic (positively charged) aminoacids such as arginine, histidine, and lysine; (3) neutral polar aminoacids such as glycine, serine, threonine, cyteine, cystine, tyrosine,asparagine, and glutamine; (4) neutral nonpolar (hydrophobic) aminoacids such as alanine, leucine, isoleucine, valine, proline,phenylalanine, tryptophan, and methionine.

[0195] Conservative amino acid changes within a fundamental polypeptidesequence can be made by substituting one amino acid within one of thesegroups with another amino acid within the same group. The encodingnucleotide sequence (gene, plasmid DNA, cDNA, or synthetic DNA) willthus have corresponding base substitutions, permitting it to encodebiologically functional equivalent forms of an AMPA transacylase.

[0196] The following examples describe preferred embodiments of theinvention. Other embodiments within the scope of the claims herein willbe apparent to one skilled in the art of endeavor from consideration ofthe specification or practice of the invention as disclosed herein. Itis intended that the specification, together with the examples, beconsidered exemplary only, with the scope and spirit of the inventionbeing indicated by the claims which follow the examples. In the examplesall percentages are given on a weight basis unless otherwise indicated.

EXAMPLES Example 1

[0197] This example illustrates the growth inhibitory effects ofN-aminomethyl phosphonic acid (AMPA) on plant callus tissue, and thelack of inhibition of N-acetyl-arminomethyl phosphonic acid on plantcallus tissue in in vitro culture conditions.

[0198] Certain recombinant plant species which express a bacterial GOXgene, and which were also exposed to glyphosate, can exhibit phytotoxiceffects manifested through such symptoms as chlorosis, flowerabscission, and reduced fertility. The basis for these symptoms had notpreviously been determined. Previous studies had indicated that plantsexpressing GOX metabolized glyphosate to AMPA and glyoxylate (U.S. Pat.No. 5,463,175). Glyoxylate is readily metabolized by plants, howeverAMPA persists in plant tissues and may be the cause of phytotoxiceffects such as chlorosis, stunting, or other undesireable effects. Ithad previously been shown that Achromobacter species LBAA was able toenzymatically modify AMPA to N-acetyl AMPA (U.S. Pat. No. 5,463,175).The Achromobacter data. coupled with the plant phytotoxicity data,indicated that N-acylation of AMPA in planta may provide effectiverelief from chlorosis and other undesireable effects. Thus, tobaccocallus tissue was exposed to AMPA and to N-acetyl AMPA in order todetermine if either of these compounds exhibited cytotoxic effectssimilar to those observed in plants expressing GOX and exposed toglyphosate.

[0199] Tobacco callus was generated from leaf pieces of wild typeNicotiana tabacum cv. “Samsun” tobacco on MS104 plates (MS salts 4.3g/l, sucrose 30 g/l, B5 vitamins 500X 2 ml/l, NAA 0.1 mg/l, and BactoAgar 1.0 mg/l). Callus tissue was applied to plates with or without AMPAand with or without N-acetyl AMPA. Plates contained AMPA or N-acetylAMPA at concentrations of 0.1 mM or 0.4 mM. Plates were incubated for upto three weeks and monitored periodically.

[0200] Callus tissue on control plates containing no AMPA or N-acetylAMPA grew at normal rates, regenerating roots and shoots as expected.Callus tissue in the presence of AMPA was severely inhibited. No growthwas observed, showing the phytotoxic effect of AMPA at theseconcentrations. Callus tissue on plates containing N-acetyl AMPA was notinhibited, and formed roots and shoots similar to control callus tissuegrowth. This result indicated that AMPA, as a byproduct of GOX mediatedmetabolism of glyphosate, could be responsible for the observedphototoxicity in plants. This result also indicated the possibility ofan improved method for selecting plants from genetically transformedcallus tissue, as well as a possible method for enhancing glyphosateherbicide resistance.

Example 2

[0201] This example illustrates that degradation of glyphosate by GOXenzyme hydrolysis in the bacterium Achromobacter sp. strain LBAA resultsin the production of AMPA and N-acetyl AMPA.

[0202] It has been previously shown that GOX mediated glyphosatedegradation produced glyoxylate and AMPA (Barry et al., U.S. Pat. No.5,463,175). Achromobacter sp. strain LBAA was also shown to produce AMPAand glyoxylate as a result of glyphosate degradation. The glyphosatedegradation pathway was characterized in resting cells ofglyphosate-grown Achromobacter sp. strain LBAA according to thefollowing procedure. Cells from a 100 ml culture of LBAA, grown in DF3Smedium containing glucose, gluconate and citrate as carbon sources andwith thiamine and Yeast Extract (0.01%) to supply trace requirements andwith glyphosate at 0.2 mM as a phosphorous source, were harvested at acell density of 200 Klett units, washed twice with 20 ml of DF3S mediumand the equivalent of 20 ml of cells were resuspended in 100 μl of thesame medium containing [¹⁴C]glyphosate (2.5 ml of 52 mCi/mmol, Amersham;CFA.745). The cell mix was incubated at 30° C. with shaking and 20 mlsamples were withdrawn at various intervals. The samples werecentrifuged to separate the cells from the broth supernatant. Both thesupernatant and cell pellets were analyzed by HPLC.

[0203] Samples prepared in this way were analyzed by strong anionexchange (SAX) HPLC with radioisotope label detection to determine theirlevels of [¹⁴C]-AMPA and N-acetyl-[¹⁴C]-AMPA. Samples were injectedusing a Waters WISP autoinjector. Chromatographic profiles andquantitative data were collected using MACS2, Monsanto's automatedchromatography data collection system. A Spherisorb S5 SAX, 250 mm×10 mmcolumn, or an Alltech 5 micron, 250 mm×10 mm SAX column was used for theanalyses. Solvents used were designated as solution A and solution B.Solution A contained 0.005M KH₂PO₄ adjusted to pH 2.0 with H₃PO₄ in 4%methanol. Solution B contained 0.10 M KH₂PO₄ adjusted to pH 2.0 withH₃PO₄ in 4% methanol. Each sample run time consisted of a step gradientprogram with an eluent flow rate of 3 ml per minute and a scintillationfluid (tradename ATOMFLOW, No. NEN-995 obtained from PackardInstruments) flow rate of 9 ml per minute. The HPLC solvent profile fordistinguishing [¹⁴C]-AMPA from N-acetyl-[¹⁴C]-AMPA in each sampleanalyzed was represented by 100% solvent A at times zero through 5minutes, then solvent B at 100% at time 5 minutes through 15 minutes,then 100% solvent A through 20 minutes at which time the column isprepared to receive another sample.

[0204] Cell pellets were first resuspended in DF3S medium made acidic byaddition of 0.65N HCl, boiled for 5 minutes, then centrifuged briefly toprovide a solution phase for HPLC analysis. Supernatants were treatedsimilarly prior to HPLC analysis. An acidified glyphosate control wasalso subjected to HPLC analysis, and the glyphosate retention time (RT)was determined to be 10.8 minutes. The amount of radioactivity in theglyphosate peak remaining in the supernatant after two hours incubationhad decreased to about 33% of the initial levels, indicating that theglyphosate was extensively metabolized. About 3% of the glyphosate wasfound to be within the cell. Material co-eluting with the methylaminestandard with an RT of 6 minutes accounted for about 5% of the initialamount of radioactivity in the supernatant and for about 1.5% of theinitial amount of radioactivity identified in the cell contents.

[0205] The GOX mediated glyphosate degradation pathway was elucidatedfurther in a subsequent experiment where the metabolism of [¹⁴C]AMPA wascompared to that of [¹⁴C]glyphosate as indicated above in resting cellsharvested at 165 Klett units and resuspended at the equivalent of 15 mlcells per 100 ml DF3S medium. The samples were analyzed by HPLC andconsisted of whole cultures acidified and treated as described above.Cultures exposed to [¹⁴C]glyphosate for two hours were found to have 25%of the label in the methylamine/N-acetyl-methylamine peak with aretention time of 14.7 minutes, 12.5% as AMPA with a retention time of 6minutes, 30% in a peak with a retention time of 13.2 minutes, and 30% asglyphosate with a retention time of 10.8 minutes. Analysis of culturesexposed to [¹⁴C]-AMPA for two hours indicated that 15% of the label wasfound as N-acetyl-methylamine/methylamine, 59% as AMPA, and 18% in the13.2 minute peak. The material eluting at 13.2 minutes was identified asN-acetyl-AMPA by negative ion electrospray mass spectrometry. The resultshowed strong ions at m/e 152 and m/e 154, as expected for thiscompound, which has a molecular weight of 153 Daltons. The m/e 154 ionwas due to the isotopic ¹⁴C atom. N-acetyl-methyl-[¹⁴C]-AMPA arises fromN-methyl-[¹⁴C]-AMPA, which is a known impurity in preparations of[¹⁴C]-AMPA.

[0206] These data indicated that the glyphosate degradation pathway inAchromobacter strain LBAA proceeds from hydrolysis of glyphosate toAMPA, which is then converted to the products methylamine presumablythrough a dephosphorylation step, and N-acetyl-AMPA presumably throughsome previously unknown transacylation step. A small amount ofN-acetyl-AMPA is then converted to N-acetyl-methylamine. A similaracylation step has been inferred from the products identified in E. coliwhen aminomethylphosphonates are utilized as sole sources of phosphate(Avila et al., 1987).

Example 3

[0207] This example illustrates the identification of an AMPAacyltransferase activity in E. coli.

[0208] Avila et al. (1987) identified dephosphorylated biodegradationproducts from the metabolism of a variety of aminophosphonate substratesused as sole phosphate sources in vivo in E. coli while studying C—Pbond scission. Their studies indicated that AMPA was not a substrate foracylation in E. coli K-12. In addition, Avila et al. were interested inthe effect of N-linked chemical substitutions on C—P bond scission ofphosphonates in E. coli, and identified N-acetylated products derivedfrom the metabolism of some aminophosphonates. Avila et al. alsodemonstrated that ‘wild type’ E. coli K12 strains, unlike wild type E.coli B strains, are unable to use phosphonates as a source of phosphate.Thus, in consideration of the phytotoxic effects of AMPA on callustissue as shown in Example 1 and the generation of AMPA from GOXmediated glyphosate degradation as shown in Example 2, the E. coli datain Avila et al. indicated that there may be an enzyme or pathway presentin some bacterial species which is capable of convertingaminomethylphosphonate (AMPA) to N-acetyl-AMPA. An enzyme or pathwaywith those characteristics would, if expressed in plants, confer asignificant advantage to plants expressing GOX when treated withglyphosate.

[0209] To test this, an E. coli K-12 strain adapted for growth on AMPAwas grown on low phosphate containing medium in order to obtain celllysates to be assayed for the presence of an enzyme capable of AMPAN-acylation. The phn (mpu) operon is cryptic in E. coli K-12 due to an 8base pair insertion which causes a frameshift mutation in the phnE gene.The frameshift inactivates PhnE and creates a polar effect ontranslation of other genes downstream of phnE within the operon,resulting in the inability of such mutants to use phosphonates asphosphate sources (Makino et al., J. Bacteriol. 173:2665-2672, 1991).Selection of a spontaneously derived mutation restores the function ofthe phn operon (phn+ or mpu+). Thus, K-12 strains adapted for growth onAMPA, methyl-phosphonate, or ethyl-phosphonate contain such effectivespontaneously derived mutations.

[0210] Briefly, an aliquot of a fresh L-broth culture of E. coli K-12strain JM101 (mpu−) was plated onto MOPS (Neidhardt et al., 1974)complete agar medium containing amino acids at 25 mg/ml, vitamin B1[thiamine] at 10 mg/ml, 0.2% glucose, and 1.5% DIFCO “Purified” agaralong with aminomethylphosphonate (AMPA; 0.2 mM; Sigma Chemical Co., St.Louis, Mo.) as the sole phosphate source, and incubated at 37° C. forthree days. Colonies arising on this media were picked and streaked ontoMOPS complete agar containing either AMPA or methylphosphonate (Alfa) asthe sole phosphate source. One colony, designated E. coli JM101 mpu+,was chosen from those that grew equally and uniformly on bothphosphonate containing media, and was further designated as E. colistrain GB993.

[0211] The phn operon is induced when E. coli is grown in media lackingor limited in a phosphate source. Therefore, E. coli GB993 was comparedto the parental JM101 strain when grown in MOPS minimal media. GB993 andits mpu− parent strain, JM101, were grown under identical conditions,varying only the amount of phosphate available or supplemented withAMPA. 50 ml cultures were grown in duplicate in 250 mlsidearm-Erlenmeyer flasks with continuous shaking at 37° C. in MOPSmedium (5 mls of 10×MOPS salts, 0.5 ml 1 mg/ml thiamin, 0.5 ml 20%glucose, to 50 mls with dH₂O) containing 0.1 or 5 mM phosphate, or 0.1mM phosphate supplemented with approximately 0.2 mM AMPA, pH 7.0. Thecultures were generally grown to about 220 Klett units and the cellswere pelleted by centrifugation, resuspended in 1.5 mls of 10 mM Tris/1mM DTT, and lysed with two passes through a French press at 1,000 psi.Lysates were centrifuged to remove debris and the supernatant passedthrough a G-50 column equilibrated with 50 mM Tris pH 7.0. Table 2 showsthe results of cell cultures grown in this manner. TABLE 2 Effects ofPhosphate Substrate on Cell Growth Strain JM101 JM101 JM101 GB993 GB993GB993 0.1 mM 5 mM 0.2 mM 0.1 mM 5 mM 0.2 mM Phosphate Phosphate AMPAPhosphate Phosphate AMPA Growth Period (hrs)  48  29 54  48  29  54Harvest Density (Klett 155 240 — 140 244 185 Units)

[0212] An HPLC assay was used to determine the presence or absence ofany AMPA acyltransferase activity in the media and cell lysates. Theassay monitors the conversion of [¹⁴C]AMPA to N-acetyl-[¹⁴C] AMPA.Generally, 100 μl of a 2×assay solution consisting of 16.5 mgacetyl-CoA, 250 μl of 2M Tris, pH 7.5, 4.5 mls dH₂O and [¹⁴C]AMPA (30mM) was mixed with 25-75 μl of lysate and 1 μl each of 0.5 M MgCl₂ andMnCl₂, and brought to 200 μl with dH₂O. The assay was incubated for 30minutes at 37° C., and quenched with 200 μl 90-100 mM NaOAc (sodiumacetate) pH 4.4 in ethanol and then analyzed immediately by HPLC asdescribed above, or stored at −20° C. Only GB993 lysate samples derivedfrom cultures grown in the presence of AMPA or 0.1 mM phosphatesupplemented media demonstrated appreciable AMPA acyltransferaseactivity. This result indicated that a gene encoding an acyltransferaseenzyme capable of AMPA N-acylation was present in GB993 and wasregulated for expression when grown under low phosphate conditions.Thus, the coding sequence for the enzymatic activity appears to be partof the pho regulon and may reside in the phn operon.

Example 4

[0213] This example illustrates the identification of an E. coli phnoperon gene encoding an enzyme capable of AMPA acylation.

[0214] Example 3 indicated that the AMPA acyltransferase activityobserved in lysates of E. coli may be encoded by a gene in the phnoperon. The entire phn operon in E. coli B and in E. coli K-12 haspreviously been cloned and sequenced B (Wanner et al., Chen et al.). TheE. coli K-12 phn operon DNA sequence has been shown to be identical tothe published DNA sequence of the phn operon from E. coli B with theexception of an eight base pair insertion in the phnE gene (Wanner etal). Clones containing various amounts of the phn operon genes fromeither bacterial genetic background are readily available (Wanner etal., Chen et al., Dr. J. W. Frost at Purdue University). Plasmidscontaining differing amounts of the JM101 phn operon DNA were used totransform JM101(mpu−) in order to test for a plasmid localized phn genethat, when expressed, confers upon JM101 the ability to utilize AMPA asa sole phosphate source.

[0215] A plasmid obtained from J. Frost (Dr. J. W. Frost, Department ofChemistry, Purdue University, West Lafayette, Ind. 47907), designatedherein as pF, contains an E. coli K-12 8 kb EcoRI fragment which encodesthe phn operon genes phnG through phnQ. A single NcoI site is present atthe 5′ end of the phng coding region. Plasmid pF was digested with EcoRIand NcoI, releasing a 2 kb NcoI-EcoRI fragment containing the genes phnGthrough phnI, and a second NcoI-EcoRI fragment about 6 kb in lengthcontaining the genes phnJ through phnQ. Each fragment was gel purifiedand ligated into a cloning and expression vector in an orientation whichwould allow for expression of the phn operon genes present within eachof the NcoI-EcoRI fragments from a plasmid borne inducible promoter. The2 kb fragment was inserted into the NcoI-EcoRI sites within the vectorpMON7258, a positive selection cloning vector identical to pUC118 withthe exception of polylinker dormain (Viera et al., Methods Enzymol.153:3, 1987), the resulting plasmid being designated as p58-1. Theorientation of the 2 kb fragment in p58-1 allows for the expression ofthe phnG-phnI genes from the lac promoter within the vector. The 6 kbEcoRI-NcoI fragment was inserted into the NcoI and EcoRI sites in asimilar positive selection vector, pMON7259, producing the plasmiddesignated as pMON17195. pMON7259 is identical to pUC119 except for thepolylinker domain, which contains a multiple cloning site opposite inorientation to that within pMON7258, and which also allows forexpression of the phnJ-phnQ genes from a lac promoter. p58-1 andpMON7259 were transformed into E. coli K 12 (mpu−) strain JM101, andmaintained with ampicillin antibiotic resistance selection. pMON7259 andpF were also transformed into JM101 as negative and positive controls,respectively.

[0216] Cultures of each transformant were grown overnight in M9 liquidbroth media supplemented with 2% casamino acids, thiamine, and 0.2%glucose with shaking at 37° C., and then diluted 1:50 into 50 ml offresh pre-warmed media of the same composition in a 250 ml side-armedErlenmeyer flask. Cultures were incubated with shaking at 37° C. untilreaching a cell density of about 80-100 Klett Units as measured on aKlett-Summerson spectrophotometer through a #2 green filter. Expressionfrom the plasmid lac promoter was induced by the addition of 100microliters of 500 mM IPTG so that the final IPTG concentration wasabout 1 mM. The induction phase growth period was allowed to progressfor two hours. Table 3 shows the cell density profile of each culturefrom 1:50 dilution through the two hour induction period. TABLE 3Induction Profile of JM101 Cultures Harboring Various phn PlasmidsCulture/ Plasmid IPTG I₀ I₁ I₂ pMON7259 + 13 75 222 p58-1 + 15 70 212pMON17195 + 15 90 220 pF + 17 97 290 pF − 15 − 260

[0217] The cells in each culture were harvested by centrifugation at10,000 rpm for 10 minutes at 4° C. in a Beckmnan J2 centrifuge. The cellpellet was washed one time in ice cold 154 mM NaCl solution, and thenresuspended in 1.5 ml extraction buffer (50 mM Tris-HCl pH 7.5, 1 mMDTT, 50 mM Tris-HCl pH 7.5). Cell suspensions were ruptured with twopasses through a French Press at 1000 psi. The resulting lysate wascentrifuged for 15 minutes at 14,000 rpm at 4° C. in an EPPENDORF™ model5402 microcentrifuge in order to remove debris. Each cleared lysate wastransferred to a fresh pre-chilled tube and the volume of the extractwas adjusted to 2.5 ml with 50 mM Tris-HCl pH 7.5. A PD10 column wasequilibrated with 25 ml 50 mM Tris-HCl, pH 7.5 and then each sample wasapplied to the desalting column. Each eluted sample was adjusted to 3.5ml with 50 mM Tris-HCl, pH 7.5. Each sample was distributed to assaytubes and mixed with reagents in order to assay for the presence of AMPAacyltransferase activity as shown in Table 4. TABLE 4 Assay Conditionsfor Bacterial Lysates Expressing phn Genes 2X Assay Extract 50 mM TrisMix Total Sample IPTG Volume* Volume* Volume* Volume* pMON7259 +  25 75100 200 pMON7259 + 100  0 100 200 p58-1 +  25 75 100 200 p58-1 + 100  0100 200 pMON17195 +  25 75 100 200 pMON17195 + 100  0 100 200 pF +  2575 100 200 pF + 100  0 100 200 pF −  25 75 100 200 pF − 100  0 100 200 −na  0 100  100 200

[0218] Each mixture was incubated at 37° C. for 30 minutes, and quenchedwith an equal volume (200 microliters) of 90-100 mM NaOAc (sodiumacetate), pH 4.4 in ethanol and if not analyzed immediately by HPLC asdescribed above, then stored overnight at −20° C. Unused portions ofeach lysate were stored either at 4° C., or mixed with glycerol to 10%by volume, and stored at −20° C.

[0219] Samples of each lysate subjected to the AMPA transacylase assaywere analyzed by HPLC for the presence of [¹⁴C]AMPA and acylated[¹⁴C]AMPA, as described above. The results are shown in Table 5. TABLE 5HPLC Analysis of Bacterial Lysate Conversion of AMPA to Acetyl-AMPASample % Acetyl AMPA % AMPA pMON7259 no data no data pMON7259  8 92p58-1  5 95 p58-1 13 87 pMON17195 100   0 pMON17195 100   0 pF 61 39 pF97  3 pF 52 48 pF 90 10 — — 100 

[0220] This data indicated that the plasmid containing the 6 kbNcoI-EcoRI fragment isolated from pF in pMON17195 contained one or moregenes which, upon IPTG induction of the lac promoter in an mpu− strainof E coli, elicited the production of an acyltransferase activitycapable of converting all of the [¹⁴C]AMPA available in the assay mix toacetyl-[¹⁴C]AMPA. The gene or genes required for AMPA N-acylation werefurther defined by restriction deletion analysis.

[0221] Plasmids containing various segments of the phn operon fromeither E. coli B or E. coli K-12 were constructed to further delineatethe nature of the phn operon gene or genes involved in conferring AMPAacyltransferase activity when expressed in an mpu− E. coli JM101.pMON7333 contains the pMON17195 equivalent E. coli DNA insertion, but inpUC119, and is a single E. coli B strain HindIII fragment containing thewild type phn operon genes phnG through phnQ. pMON15020 was constructedby cloning a 5,713 base pair NcoI to EcoRI E. coli B DNA fragment frompMON7333 into pMON7259, and contains the genes phnJ through phnQ.pMON15022 was constructed by inserting a 1,686 base pair EcoRI to SalIfragment from pMON17195 into the positive selection cloning andexpression vector pBlueScriptSP (Invitrogen), which contains the E. coliK-12 genes phnO, P and Q. pMON15023 was constructed by deleting an 1,820base pair SalI fragment from pMON17195, leaving behind the E. coli K-12phn operon genes phnJ and phnK, the 5′end of phnL, and all of phnO, Pand Q.

[0222] The plasmids pMON17195, pMON15020, pMON15022, pMON15023, andpMON7259 were transformed into the mpu− E. coli K-12 strain JM101 andwere maintained by ampicillin antibiotic selection. Overnight culturesof each of these transformants were grown with antibiotic selection andwere diluted 1:50 into fresh M9 media as described above, and incubatedat 37° C. with shaking in 250 ml sidearm-Erlenmeyer flasks to a celldensity of about 100 Klett units. Each culture was induced with IPTG asin example 3, and incubated for two additional hours with shaking. Thecells were harvested by centrifugation in a Beckman J2 centrifuge at4,000 RPM for 10 minutes at 4° C. Cell pellets were washed once with 50ml of 154 mM NaCl, and stored at −20° C.

[0223] Cell pellets were resuspended in 1.5 ml Extraction Buffer as inexample 3 and ruptured by two passes through a French Press at 1000 psi.The ruptured cell suspensions were centrifuged in an Eppindorfmicrocentrifuge Model 5402 for 15 minutes at 14,000 rpm and at 4° C. Thecleared lysates were decanted into new tubes pre-chilled on ice, and thetotal volume was adjusted to 2.5 ml with addition of Extraction Buffer.These samples were desalted over a PD10 column pre-equilibrated with 25ml of 50 MM Tris-HCl, pH 7.5, and eluted with 3.5 ml of 50 mM Tris HClpH 7.5. Samples were then subjected to an AMPA acylation assay asdescribed above, incubated for 30 minutes at 37° C., and quenched with200 microliters of 90.9 mM NaOAc pH 4.4 The volumes of each sample usedin the assay are noted in Table 6. All volumes represent microliters ofeach solution used. TABLE 6 Assay Conditions for Bacterial LysatesExpressing phn Genes from Plasmids Plasmid Extract 50 mM Tris 2X AssayMix Total Volume — — 100  100 200 pMON 17195  25 75 100 200 pMON 17195100 — 100 200 pMON 15020  75 75 100 200 pMON 15020 100 — 100 200 pMON15022  75 75 100 200 pMON 15022 100 — 100 200 pMON 15023  75 75 100 200pMON 15023 100 — 100 200 pMON 7259  75 75 100 200 pMON 7259 100 — 100200

[0224] Quenched samples were subjected to HPLC analysis as describedabove. Table 7 illustrates the results of HPLC analysis of each sample,indicating the relative amount of [¹⁴C] AMPA or acetyl-[¹⁴C]AMPA as apercentage of the total amount of [¹⁴C] in both peaks combined. TABLE 7HPLC Analysis of Bacterial Lysate [¹⁴C]-AMPA Conversion toAcetyl-[¹⁴C]-AMPA Extract % [¹⁴C]- % Acetyl- Total % Sample Volume AMPA[¹⁴C]-AMPA [¹⁴C] — — 100  — 100 pMON17195  25 66  34 100 pMON17195 10026  74 100 pMON15020  75 — 100 100 pMON15020 100 — 100 100 pMON15022  75— 100 100 pMON15022 100 — 100 100 pMON15023  75 — 100 100 pMON15023 100— 100 100 pMON 7259  75 87  13 100 pMON 7259 100 72  28 100

[0225] The data in Table 7 indicates that AMPA acylation activity isderived from the phn operon open reading frames consisting of phnO,phnP, and phnQ, which are the only phn genes present in pMON15022. Otherplasmids conferring AMPA acylation activity upon induction alsocontained at least the phnO, P, and Q genes, providing strong evidencethat the observed activity was the result of one or more of these geneproducts. Therefore, additional plasmids were constructed based on thephnO, P, and Q gene sequences in order to determine which gene or geneswere required for the acylation function.

[0226] Bacterial acylase, transacylase, and acyltransferase genes havebeen known in the literature for some time. Most are small 15-25 K Daproteins. Therefore, on the basis of size comparison, only the phnO andphnQ gene products would fall into this category. However, based onsimilarity comparisons with other proteins in the GENBANK, SWISSPROT,and EMBL databases, the predicted phnO gene product appeared to mostclosely resemble other proteins having acylase activity. For example,the E. coli PhnO protein aligned well with a gentamicinacetyltransferase-3-I described in Wohlleben et al. (Mol. Gen. Genet.217:202-208, 1989). pMON15020 containing the E. coli B phn operon genesphnJ through phnP on a single 6.0 kb NcoI-EcoRI fragment was digestedwith SalI and EcoRI to release a 2.0 kb fragment containing the phnO, Pand Q genes. This 2 kb fragment was excised and purified from a 0.7% TAEAgarose gel, treated with T4 DNA polymerase to excise the 3′ overhangingends, then with Klenow and deoxynucleotide triphosphates (dXTP's) toprovide blunt ends, and then ligated into the EcoRV site ofpBlueScriptSP to produce plasmid pMON15024. pMON15024 was digested withNdeI and EcoRI, deleting a 1200 base pair fragment containing most ofthe phnP and all of the phnQ coding sequences. The remaining pMON15024plasmid fragment still containing the phnO gene was treated with Klenowfragment DNA polymerase in the presence of dideoxynucleotides accordingto the manufacturer's instructions in order to fill in the 3′ endsexposed by restriction enzyme digestion, then ligated together toproduce the plasmid pMON15027. pMON15027 contains only the phnO geneflanked 3′ by a small portion of phnP. The 1200 base pair NdeI to EcoRIfragment obtained from pMON15024 was cloned into pMON2123 to producepMON15026, which contains the 3′ two thirds of the phnP gene flanked 3′by phnQ. Plasmids pMON15024, 15026, and 15027 were introduced into mpu−JM101, and cell lysates of transformants were analyzed as above aftergrowth and induction for the presence of AMPA acyltransferase activity.Only pMON15024 and pMON15027 exhibited acyltransferase activity,indicating that the phnO gene product was responsible for AMPAacylation.

[0227] A DNA fragment containing only the phnO gene with convenientflanking restriction endonuclease sites for use in further cloningmanipulations was produced using thermal cycling methods. Syntheticoligonucleotide primers were synthesized by Midland Certified Reagents,Co. (Midland Tex.) based on the published phnO gene and flankingsequence in order to amplify the phnO gene (Chen et al., J. Biol. Chem.256: 4461-4471, 1990). The sequence AAACACCATGGCTGCTTGTG (SEQ ID NO: 5),designated AATPCR6, represents a synthetic oligonucleotide which ishomologous to the template strand of the phnO gene. The 5′ adenosineresidue of SEQ ID NO: 5 corresponds to base pair 13,955 of the publishedphn operon sequence, immediately 5′ of the phnO ATG initiation codon atposition 13,962-13,964 (Chen et al., J. Biol. Chem. 256: 4461-4471,1990). SEQ ID NO: 5 incorporates a single base pair mismatch from thepublished phnO sequence at position 13,965 represented by a C to Ginversion, which generates an alanine codon in place of a proline codonat position 2 and also creates a unique NcoI restriction site spanningthe ATG initiation codon. The sequenceGTGACGAATTCGAGCTCATTACAGCGCCTTGGTGA (SEQ ID NO: 6), designated AATPCR7,represents a synthetic oligonucleotide which is homologous to the codingstrand of the phnO gene. The 3′ adenosine residue of SEQ ID NO: 6corresponds to base pair 14,380 of the published phn operon (Chen etal., J. Biol. Chem. 256: 4461-4471, 1990). The thymidine at positionnumber nineteen of SEQ ID NO: 6 corresponds to the adenosine at position14,396 of the published phnO sequence (Chen et al.). A portion of SEQ IDNO: 6 overlaps the native phnO termination codon, introduces a second inframe termination codon immediately 3′ of and adjacent to the nativetermination codon, and also introduces unique EcoRI and Sacl restrictionsites 3′ of these termination codons.

[0228] pMON15024 was used as a template for amplification of the phnOgene in a standard thermal amplification reaction. Briefly, a 100microliter reaction sample was prepared which contained 0.1 ng templateDNA, reaction buffer, 200 pM each primer, 200 mM dNTP, 1.25 U Taq DNApolymerase and was overlayed with mineral oil. This reaction sample wassubjected to thirty five cycles at 94° C. for one minute, 50° C. for twominutes, and 72° C. for three minutes which resulted in theamplification of a 459 base pair DNA product as determined by analysisof five microliters of the reaction sample on a ethidium bromide stained0.7% TAE agarose gel. A 444 base pair product was purified usingstandard methods from a 1% TAE agarose gel after digestion of a sampleof the 459 base pair amplification product with NcoI and EcoRIrestriction endonucleases. The 444 base pair product was ligated intocompatible sites in pMON7259 to generate pMON15028. Cell lysatesprepared as above from IPTG induced cultures of JM101 containingpMON15028 were analyzed for the presence of AMPA acyltransferaseactivity and compared to cultures containing pMON15027. The results wereindistinguishable, thus confirming that phnO encoded an enzyme capableof AMPA acylation. In addition, this result indicated that the P2Amutation in the protein, which was introduced into the gene codingsequence as a result of thermal amplification using the AATPCR6oligonucleotide primer (SEQ ID NO: 5), was without effect on theacyltransferase activity of the resulting PhnO protein when expressed inE. coli.

Example 5

[0229] This example illustrates the production of polyclonal antibodiesdirected to the PhnO peptide.

[0230] Further studies of the phnO gene product required the use ofantibodies directed to the PhnO protein. Therefore, PhnO wasoverproduced in E. coli JM101 for for use as an immunogen in stimulatingthe production of antibodies upon injection into a goat. The phnO genecontaining the P2A mutation in plasmid pMON15028 was introduced intoplasmid pMON17061 on an NcoI to EcoRI DNA fragment, producing pMON15032.phnO expression in pMON15032 is under the control of the E. coli recApromoter adjacent to the bacteriophage T7 gene 10L ribosome bindingsequence. Cells were grown to mid log phase and induced by addition ofnalidixic acid to the culture to approximately 50 parts per million,from a stock solution of 50 mg nalidixic acid powder dissolved in 1 ml0.1 N NaOH. The culture was maintained under inducing conditions fortwelve hours at 37° C. Cells were harvested as described in example 3,and sonicated in phosphate buffered saline. About 23% of the totalsoluble protein in the induced E. coli lysates was determined to be PhnOand approximately 60% of the total PhnO protein was released into thesoluble phase as judged by SDS-PAGE and Coomassie blue staining. Theprotein was further purified by preparative SDS-PAGE providing asufficient quantity of PhnO for use in producing antibody which binds toor reacts antigenically with PhnO or related AMPA transacylase proteins.Briefly, the PhnO protein was separated by size from other proteins in a15% SDS-PAGE gel. A gel slice containing the PhnO protein was excised,weighed, and homogenized using a polytron in a volume of phosphatebuffered saline (PBS, pH 7.0) equal to the mass of the gel slice. Thehomogenate was mixed with an equal volume of complete Freund's mediauntil a colloidal mixture was obtained. An 8-ml inoculum of this mixturewas used for the first injection into a goat. Two weeks post-injection,a 50-ml bleed was collected and serum was separated from blood solids bycentrifugation. A booster injection of gel purified PhnO protein wasadministered in a colloidal mixture of 50% incomplete Freund's adjuvantat four weeks, and at six weeks a second bleed was obtained.

[0231] The serum from the second bleed was used to screen for thepresence of sufficient antibody titers specific for PhnO protein.Extracts from JM101 cells containing pMON15032 were subjected to westernblot analysis. The concentration of protein in the extract wasdetermined to be about 55 mg/ml by Bradford assay, and a prior Coomassiestained gel using this same extract was subjected to a densitometer scanwhich indicated that about 23% of the total cell protein was PhnO. Theextract was desalted over a PD10 column, eluted with 10 mM Tris pH 7.5,and diluted with an equal volume of 2×SDS sample buffer. Serialdilutions were prepared using 1× sample buffer and loaded into wells ofa 15% SDS PAGE gel. Additional samples were mixed with a tobacco leafprotein extract containing 10 additional micrograms of protein per lanein addition to the E. coli PhnO extracts. The tobacco leaf proteinextracts were used to screen for the presence of cross reactive antibodyto plant proteins. Proteins were separated according to size byelectrophoresis at 7.5 mA constant for fourteen hours at 4° C., and thegel was electroblotted onto a MSI 0.45 micron nitrocellulose filter at0.5 Ampere in Tris-Glycine transfer buffer for one hour. The membranewas then blocked with TBST (Tris, BSA, NaCl, Tween-20, Short Protocolsin Molecular Biology, 3rd Ed., Wiley and Sons, Pub.) for two hours atroom temperature, incubated forty-five minutes with a 1:500 dilution ofthe second bleed serum at room temperature, washed two times in TBST,incubated another forty-five minutes with alkaline phosphataseconjugated rabbit anti-goat IgG (Boehringer Mannheim Biochemicals,Inc.), washed three times with TBST and one time with alkalinephosphatase buffer, and finally incubated for two and one half minuteswith a standard color development solution containing NBT and BCIP. Thereaction was terminated by washing the membrane with ample quantities ofdistilled water. The antibody was able to detect PhnO protein in aslittle as 50 nanograms of E. coli extract independent of the presence ofadditional plant proteins in one half of the samples. In addition, veryfew cross reactive bands were detected in either set of samples,indicating that the serum sample contains very little IgG which crossreacts with either E. coli or tobacco plant proteins when tested usingthis western blot method.

[0232] An alternative source for generating antibody which is capable ofspecific binding to or reacting antigenically with PhnO protein was alsoutilized. A phnO gene was placed into a commercial vector (Invitrogen)containing a metal binding amino acid coding sequence (His6) upstream ofand in frame with the phnO coding sequence. The His6-phnO DNA sequencewas inserted into the E. coli expression vector pMON6235 on an NcoI toEcoRI fragment, under the control of an E. coli arabinose operon araBADpromoter, producing plasmid pMON32909. His6-PhnO protein was producedupon arabinose induction of E. coli W3110 cells containing pMON32909,and purified over a metal affinity column according to themanufacturers' instructions.

[0233] His-tagged purified His6-PhnO protein standard was injected into6 New Zealand White rabbits using an immunization procedure similar tothat used for the goat, described above. Antiserum raised in theserabbits was also shown to be specific for binding PhnO protein andnon-cross reactive with other E. coli bacterial or tobacco plantproteins.

Example 6

[0234] This example illustrates properties of an AMPA transacylaseenzyme using aminomethylphosphonate and acetyl-CoA as substrates in anenzyme assay as measured by endpoint kinetic analysis.

[0235] The apparent Km (Km) and Vmax (Vmax) of PhnO enzyme weredetermined for the substrates aminomethlyphosphonate and acetyl-CoA.Determination of the PhnO Km and Vmax were made by endpoint kineticanalyses, determining the enzyme velocity in consuming each substrate atvarying substrate concentrations, and plotting the inverse of the enzymevelocity versus the inverse of the substrate concentration to produce aLineweaver-Burk plot of enzyme kinetics. The conversion of [¹⁴C]-AMPA toN-acetyl-[¹⁴C]-AMPA was monitored as in example 2, using enzyme in adesalted crude lysate of E. coli expressing phnO from pMON15032,produced as in example 4. Total protein per ml of extract was determinedby the method of Bradford which indicated approximately 22.5 ml.Densitometric scanning of Coomassie stained SDS-polyacrylamide gelsresolving PhnO protein from these lysates indicated that PhnO representsabout 23% of total protein, thus the cell extract was determined tocontain about 5.2 mg PhnO protein per ml. In a first assay to determinethe apparent K_(m) and V_(max) of PhnO for AMPA, [¹⁴C]-AMPAconcentrations ranged from 2 to 38 mM. Enzyme reactions were incubatedat 37° C. for 5 minutes and quenched with 1 volume of 100 mM sodiumacetate (NaOAc), pH 4.4, in ethanol. Samples were analyzed by HPLC todetermine the amount of [¹⁴C]-AMPA converted to N-acetyl-[¹⁴C]-AMPA. Theassay conditions and output for each set of reactions are shown in Table8. TABLE 8 PhnO Enzyme Kinetics for AMPA Substrate Sample S¹ % Turnover²Velocity³ 1/S 1/V V/S 1  200 39.5  79 1.0  0.0127 79.00 2  400 35.1 1400.5  0.0071 70.00 3  800 32.9 263 0.25  0.0038 65.75 4 1200 26.8 3220.166 0.0031 53.67 5 1600 26.2 426 0.125 0.0023 53.25 6 2000 22.1 4420.100 0.0023 44.20 7 2400 19.2 461 0.083 0.0022 38.42 8 2800 17.6 4930.071 0.0020 35.21 9 3200 17.3 554 0.063 0.0018 34.63 10  3600 14.5 5220.056 0.0019 29.00 11  4000 13.6 544 0.050 0.0018 27.20 12  6000 12.7762 0.033 0.0013 25.15 13  7600 10   760 0.026 0.0013 19.76

[0236] A Linweaver-Burk plot of the 1/V vs 1/S data from Table 8indicates that the apparent Km of PhnO for AMPA as a substrate is about9 mM, and the apparent Vmax is about 824 U/mg protein.

[0237] The apparent Km of PhnO for the substrate acetyl-CoA wasdetermined in similar experiments. After several attempts to obtain endpoint kinetics, it was determined that the turnover number was too lowto be reliable at AMPA concentrations of about 30 mM and enzyme amountsof about 1-10 ng. An alternative approach was tried using tritiumlabeled acetyl-CoA. The specific activity of the label was about 40×higher than with [¹⁴C], providing a gain in sensitivity that allowed forthe determination of the apparent Km of PhnO for Acetyl-CoA. The[³H]-acetyl-CoA (Amersham, Inc.) specific activity was 360 mCi/mg or 250μCi/ml. The transacylation mediated by PhnO from [³H]-acetyl-CoA to[³H]-acetyl-AMPA was monitored by weak anion exchange HPLCchromatography, with the retention times of acetyl-CoA and acetyl-AMPAadjusted so that these compounds were separated by about three minutes.This was accomplished by adjusting the concentration of KH₂PO₄ buffer(pH 5.5) to 40 mM with a flow rate of 1 ml per minute over an AX100 weakanion exchange column. Each sample was reacted with PhnO and 30 mM AMPAfor five minutes at 37° C. and quenched with 100 mM NaOAc pH 4.4 inethanol, then analyzed by HPLC. [³H]-acetyl-CoA substrate ranged from 25micromolar to 1.3 mM in each reaction along with about 5 ng PhnO, 50 mMTris pH 7.5, 1 mM MnCl₂, 1 mM MgCl₂, and 30 mM AMPA. Samples wereanalyzed by HPLC to determine the amounts of N-[³H]-acetyl-AMPAproduced, and [³H]-acetyl-CoA remaining. The assay conditions andresults for these reactions are shown in Table 9. TABLE 9 PhnO EnzymeKinetics for Acetyl-CoA Donor Substrate Sample No. [Acetyl-CoA]¹Velocity² 1/[S]³ 1/V⁴ V/S⁵  1  25  34 0.0400 0.0294 1.3600  2  50  660.0200 0.0152 1.3200  3  75  94 0.0133 0.0106 1.2533  4 100 125 0.01000.0080 1.2500  5 125 150 0.0080 0.0067 1.2000  6 150 173 0.0066 0.00581.1533  7 175 193 0.0057 0.0052 1.1029  8 200 219 0.0050 0.0046 1.0950 9 225 240 0.0044 0.0042 1.0667 10 250 259 0.0040 0.0039 1.0360 11 375339 0.0027 0.0030 0.9040 12 390 287 0.0026 0.0035 0.7359 13 520 3310.0019 0.0030 0.6365 14 650 352 0.0015 0.0028 0.5415 15 780 372 0.00130.0027 0.4769 16 910 397 0.0011 0.0025 0.4363 17 1040  411 0.0009 0.00240.3952 18 1170  425 0.0008 0.0024 0.3632 19 1300  434 0.0007 0.00230.3338

[0238] A Linweaver-Burk plot of the 1/V vs 1/S data from Table 9indicates that the apparent Km of PhnO for acetyl-CoA as a substrate isbetween 375-390 micromolar, and the apparent Vmax is about 824 U/mgprotein.

[0239] An approximate pH range of activity for the PhnO enzyme wasdetermined using enzyme in a crude lysate of E. coli expressing phnOfrom pMON15032. The ability of the enzyme to produce N-acetyl AMPA froma mixture containing acetyl-CoA and AMPA across a range of pH values wasdetermined. The reactions were carried out in MES/MOPS/Tricine bufferequilibrated to a pH value from 4.5 to 9.0, with actual pH valuesranging from 5.2 through 9.0. Briefly, 95 microliters of an appropriatebuffer was mixed with 100 microliters of 2×assay mix as described inexample 4, and 5 microliters of desalted E. coli lysate containingapproximately 400 ng/microliter PhnO protein. The reaction was incubatedat 37° C. for five minutes and quenched with 100 mM NaOAc pH 4.4 inethanol, and analyzed by HPLC as described in example 4. The results areshown in Table 10. TABLE 10 PhnO Enzyme pH Profile ³Velocity Buffer¹Mock ²% N-Acetyl CoA (nmole/min/ pH Reaction pH Turnover (nmole)microgram) 5.0 5.23  3.7  222  22.2 5.5 5.62  3.9  234  23.4 6.0 5.92 4.2  252  25.2 6.5 6.47 13.3  798  79.8 7.0 7.0  27.0 1620 162.0 7.57.48 32.0 1920 192.0 8.0 8.05 34.3 2058 205.8 8.5 8.46 33.5 2010 201.09.0 9.0  33.9 2034 203.4

[0240] The results indicate that optimum PhnO transacylase activityusing AMPA and acetyl-CoA as substrates is about pH 8.0. However PhnOefficiently converts AMPA to N-acetyl-AMPA using acetyl-CoA as theacetyl donor across a pH range from about 6.5 to at least 9.0.

[0241] Additional experiments were carried out with purified PhnOprotein to further characterize the scope of the enzyme's substratepreference for acyl-CoA acyl donor compounds. It has been establishedherein that at least one substrate acyl-donor or leaving group can be atwo carbon acid compound such as the acetyl-moiety in the compoundAcetyl-CoA. It was not known what range of acyl-molecules comprised ofdifferent carbon chain lengths would or could function as a leavinggroup from the acyl-CoA acyl donor when reacted with PhnO transacylaseand AMPA as the acyl-receptor molecule. Therefor, an HPLC assay similarto that described in Example 2 was developed to determine the scope ofthe enzymes' ability to transfer an acyl-group from an acyl-CoA acyldonor to [¹⁴C]-AMPA.

[0242] PhnO was purified from a one liter Luria Bertani broth culture ofE. coli JM101 expressing a recombinant phnO gene from pMON15032 afternalidixic acid induction for three hours at 37° C. Cells were harvestedby centrifugation and resuspended in 40 ml cold Tris buffer (0.1 MTris-HCl pH 8) and placed on ice. The cell suspension was brought to 1mM DTT and 0.5 mM PMSF. The suspension was lysed by 2 passages through aprechilled French pressure cell at 1,100 psi, centrifuged at 12,000 g(10,000 rpm in an Sorvall SA600 rotor) for 40 min at 4° C., then placedon ice. The cleared supernatants were poured into fresh 15 mlpolypropylene tubes. The samples were split again into two equalportions and maintained at −80° C. until used further for purificationof PhnO protein. 20 microliters of the soluble fraction was assayed forenzyme activity using the HPLC method described above in Example 2,except after terminating the assay with acid addition, the sample wasstored at −80° C. A Sephacryl S200 column was prepared according to themanufacturers' instructions and equilibrated with a solution containing20 mM Tris pH 8.0 and 0.5 mM MgCl₂. The entire total soluble extract waslayered over the top of the column bed after thawing on ice. Forty 9 mlfractions were collected from the column eluate, and thirty microlitersof each fraction was analyzed by western blot using anti-PhnO antiserumafter resolution on a 15% SDS-PAGE gel. Also, thirty microliters of eachfraction was analyzed for AMPA acyl transferase activity using themethod described in Example 2. Samples which exhibited acyl transferaseactivity and which corresponded to positive western blot data werepooled. These were represented by fractions 7 through 19 in thisexample, and were combined into a 100 ml volume, distributed into ten 10tubes each containing 10 ml volumes, and stored at −80° C. for furtheruse.

[0243] Anion exchange chromatography was used to determine the elutionpattern of PhnO away from other contaminating proteins that co-eluteduring the Sephacryl S200 fractionation. One tube from the combined PhnOpositive fractions was thawed on ice and injected into a 5/5 Mono-Qcolumn pre-equilibrated with buffers A (one liter of 20 mM Tris-HCl pH8.0 Mili-Q distilled deionized water) and B (one liter of 20 mM Tris-HClpH 8.0, 1 M NaCl). The sample containing PhnO active protein wasinjected into the column and one milliliter fractions were collected.The column was washed for five minutes with a flow rate of 1.8 ml perminute Buffer A after loading the PhnO containing sample. At fiveminutes, Buffer B was added to the flow volume at 0.5 ml per minute forfour minutes. Buffer B was ramped up to 22% of the flow volume at 10minutes, 30% at 12 minutes, 36% at 13 minutes, 41% at 14 minutes, 46% at15 minutes, 74% at 16 minutes, and 100% at 16 minutes through 22minutes, at which point Buffer B flow was terminated and Buffer A wasreinitiated at 100% to equilibrate the column. Ten microliter volumesfrom individual fractions collected from the Mono-Q column were analyzedby western blot and for transacylase activity as described in Example 2.Fractions which exhibited positive AMPA acyltransferase activity andwhich correlated with the Western blot data were pooled and maintainedas a purified protein sample. Samples of this purified PhnO protein wereused to determine enzyme's acyl donor substrate specificity.

[0244] Enzyme reactions were prepared as follows. 100 microliterreactions consisted of 50 mM Tris-HCl pH 8.0, 1 mM MgCl₂, 3 microlitersof 1.3 mM [¹⁴C]-AMPA (115,392 dpm per microliter), 0.1 mM or 1 mMacyl-CoA acyl donor, and 2.5 microliter purified enzyme sample. A assaypremix was prepared from which 45 microliters was used in each 100microliter reaction. This 45 microliter premix sample consisted of 40microliters distilled and deionized water, 2 microliters of 50 mM MgCl₂,and 3 microliters of 1.3 mM [¹⁴C]-AMPA (115,392 dpm per microliter).Reactions were initiated by mixing 40 microliters of 125 mM Tris-HCl pH8.0, 2.5 microliters protein sample and 10 microliters acyl-CoA acyldonor compound in a microcentrifuge tube at room temperature. Eachacyl-CoA acyl donor compound was prepared as a stock solution of 1 mM, 5mM or 10 mM stocks. Each tube was then mixed with 45 microliters of theassay premix containing the [¹⁴C]-AMPA receptor substrate, mixed gentlyand transferred to a 30° C. water bath for 5 minutes. Each reaction wasterminated with the addition of 4 microliters of 1M HCl, mixed byvortexing, and placed on ice or stored at −20° C. until assayed for thepresence of [¹⁴C]-AMPA or related compounds by HPLC.

[0245] HPLC analysis was carried out using a Waters 510 dual pump HPLCsystem with a 481 wavelength max UV detector and a scintillation pump, aPhenomenex PHENOSPHERE 5 micrometer 80 Å SAX-silica HPLC column (250×4.6mm, 3500 PSI max pressure), Buffer A consisting of 5 mM KH₂PO₄, 4%methanol, adjusted to pH 2.0 with H₃PO₄, and Buffer B consisting of 200mM KH₂PO₄, 4% methanol adjusted to pH 2.0 with H₃PO₄, and HAZARDAtomflow (Packard) containing 64% 1,2,4 trimethylbenzene, 7.5%sodium-dicotyl sulfosuccinate, 3.5% sodium diamylsulfosuccinate, and 6%polyoxyethylene(4)lauryl ether. HPLC gradient conditions for each sampleanalysis were similar to those described in Example 2, with minorvariations. The flow rates are provided in Table 11. TABLE 11 HPLCGradient Conditions Time Flow (min) (ml/min) % A % B Flow Rate¹  0.0 1100 0 3  2.0 1 100 0 3  5.0 1  50 50  3 15.0 1  0 100  3 17.0 1  0 100 3 17.3 1 100 0 3 21.0 1 100 0 3 21.3   0.1 100 0 0

[0246] Stock solutions of Acyl-CoA acyl donor compounds were prepared asdescribed above, and these are listed here: Na Acetyl-CoA, Lin-propionyl-CoA, Li glutaryl-CoA, Li methylmalonyl CoA, Licrotonoyl-CoA, Li isobutyryl-CoA, Na succinyl-CoA, Li tiglyl-CoA, Lin-valeryl-CoA, and Li desulfo-CoA. All compounds were obtained fromSigma Chemical Company, St. Louis, Mo. The percent activity of thepurified enzyme for transfer of the CoA associated acyl-moiety to[¹⁴C]-AMPA was determined by measuring the percentage of [¹⁴C]-AMPA HPLCchromatogram peak area converted to some other [¹⁴C]-compound, such asN-acetyl-[¹⁴C]-AMPA, with the amount of N-acetyl-[¹⁴C]-AMPA producedduring the reaction in which [¹⁴C]-AMPA and 1 mM acetyl-CoA aresubstrates for PhnO being established as the 100% reference. The resultsare shown in Table 12. TABLE 12 AMPA Transacylase Enzyme Efficiency forAcyl-CoA Acyl Donor Substrate Acyl-CoA Acyl Donor [¹⁴C]-AMPA %Conversion¹ % Activity Acetyl-CoA 0.1 mM 79.2 79.2 Acety-CoA 0.5 mM 98.798.7 Acety-CoA 1 mM 100.00 100.00 Propionyl-CoA 0.1 mM 78.2 78.2Propionyl-CoA 0.5 mM 97.8 97.8 Propionyl-CoA 1 mM 100.00 100.00Glutaryl-CoA 0.1 mM 0.81 0.81 Glutaryl-CoA 0.5 mM 0.00 0.00 Glutaryl-CoA1 mM 0.57 0.57 Methylmalonyl-CoA 0.1 mM 1.11 1.11 Methylmalonyl-CoA 0.5mM 2.08 2.08 Methylmalonyl-CoA 1 mM 2.21 2.21 Crotonoyl-CoA 0.1 mM 0.800.80 Crotonoyl-CoA 0.5 mM 0.00 0.00 Crotonoyl-CoA 1 mM 0.00 0.00Isobutyryl-CoA 0.1 mM 2.10 2.10 Isobutyryl-CoA 0.5 mM 0.20 0.20Isobutyryl-CoA 1 mM 0.00 0.00 Succinyl-CoA 0.1 mM 5.06 5.06 Succinyl-CoA0.5 mM 3.38 3.38 Succinyl-CoA 1 mM 1.56 1.56 Tiglyl-CoA 0.1 mM 0.00 0.00Tiglyl-CoA 0.5 mM 0.00 0.00 Tiglyl-CoA 1 mM 0.99 0.99 Valeryl-CoA 0.1 mM0.24 0.24 Valeryl-CoA 0.5 mM 0.00 0.00 Valeryl-CoA 1 mM 0.33 0.33Desulfo-CoA 0.1 mM 0.95 0.95 Desulfo-CoA 0.5 mM 1.25 1.25 Desulfo-CoA 1mM 0.52 0.52

[0247] These results indicate that PhnO enzyme is capable of efficientlyutilizing acyl-CoA associated compounds which have an acyl group with acarbon chain length of not more than three for transacylating AMPA.Other compounds which have a longer carbon chain length than propionyl-and which are not broad or bulky, such as methylmalonly-, isobutyryl-,and succinyl-CoA compounds are also effective acyl-CoA acyl donors, butat a lower enzyme efficiency.

Example 7

[0248] This example illustrates the in vitro expression and targeting ofan AMPA acyltransferase protein into isolated chloroplasts.

[0249] Many chloroplast-localized proteins are expressed from nucleargenes as precursors and are targeted to the chloroplast by a chloroplasttransit peptide (CTP). The CTP is removed during steps involved inimport of the targeted protein into the chloroplast. Examples of suchchloroplast proteins include the small subunit (SSU) ofribulose-1,5-bisphosphate carboxylase (RUBISCO),5-enol-pyruvylshikimate-3-phosphate (EPSPS), ferredoxin, ferredoxinoxidoreductase, the light-harvesting-complex protein I and protein II,and thioredoxin F. It has been demonstrated in vivo and in vitro thatnon-chloroplast proteins may be targeted to the chloroplast by use offusions with a CTP and that a CTP sequence is sufficient to target aprotein to the chloroplast (Della-Cioppa et al., 1987).5-enolpyruvylshikimate-3-phosphate synthetase (EPSPS) enzyme is locatedin the chloroplast and is the glyphosate target in plants. Targetingglyphosate oxidoreductase to the chloroplast has been found to providetolerance to plants to glyphosate, although GOX localized to thecytoplasm is also able to provide such tolerance. Generally, recombinantGOX enzyme is localized to the chloroplast. GOX mediated glyphosatemetabolism produces AMPA, which has been shown to be phytotoxic. It hasbeen shown herein that PhnO is capable of AMPA N-acylation and thatN-acetyl-AMPA is not phytotoxic. Therefore, it may be necessary toinactivate AMPA in plants. This assumes that AMPA acyltransferase can beexpressed in plants as an active enzyme, and that such acyltransferasesare capable of being imported into the chloroplast and retain enzymaticactivity. In view of the AMPA phytotoxicity as described in example 1,an AMPA acyltransferase gene was introduced into plant expressionvectors to test expression in plants. In addition, import ofacyltransferase into chloroplasts was also tested.

[0250] A DNA sequence encoding a chloroplast targeting peptide waslinked 5′ to and in frame with a DNA sequence encoding an AMPAacyltransferase. A DNA sequence encoding an arabidopsisribulose-1-bis-phosphate carboxylase small subunit chloroplast transitpeptide (CTP, SEQ ID NO: 9) was excised from pMON17058 using BglII andNcoI restriction endonucleases, and inserted into complementaryrestriction sites in pMON15028 to produce pMON15029, so that the CTPcoding sequence was linked 5′ to and in frame with the phnO codingsequence in pMON15028. The resulting chimeric phnO gene in pMON15029 iscapable of producing a chloroplast targeted PhnO protein. An EcoRI toBglII DNA cassette containing the CTP-PhnO coding sequence, SEQ ID NO:11, from pMON15029 was inserted into EcoRI and BamHI sites inpBlueScript KS(−) to produce pMON15036. The CTP-PhnO coding sequence inpMON15036 can be expressed in an in vitro transcription/translationsystem from a phage T3 promoter. A similar plant transient expressionplasmid, pMON15035, was constructed, but without the chloroplasttargeting sequence. An EcoRI to BglII DNA fragment containing only thephnO coding sequence was excised from pMON15028 and inserted into EcoRIand BamHI sites in pBlueScript KS(+) so that PhnO could be produced froma phage T7 promoter in an in vitro transcription/translation system. AnNcoI to EcoRI DNA sequence encoding PhnO was excised from pMON15028 andinserted into pMON17061, producing pMON15032. pMON15032 provides forexpression of phnO from an E. coli recA promoter. A BglII to EcoRI DNAfragment encoding PhnO was excised from pMON15028 and inserted intopBlueScript SK(−) to produce pMON15033. pMON15033 provides forexpression of phnO from an E. coli lac promoter. A BglII to EcoRI DNAfragment encoding CTP-PhnO was excised from pMON15029 and inserted intocompatible sites in pBlueScript SK(−), providing for expression ofchloroplast targeted PhnO protein from an E. coli lac promoter frompMON15034.

[0251] pMON15032, pMON15033, and pMON15034 were introduced into E. coliJM101. Cultures were grown and induced as described above, except thatexpression from cells containing pMON15032 was induced with addition of50 parts per million nalidixic acid in 0.1 M NaOH. Cleared lysates wereprepared from each culture and subjected to an AMPA acyltransferaseassay as described above in order to determine the presence of AMPAacyltransferase activity. All lysates contained substantial amounts ofacyltransferase activity above control levels. More importantly, theCTP-PhnO peptide (SEQ ID NO: 12) expressed from pMON15034 appeared toretain full enzymatic acyltransferase activity.

[0252] pMON15035 (PhnO) and pMON15036 (CTP-PhnO) were used in vitro togenerate [³⁵S]-methionine labeled PhnO protein for use in a chloroplastimport assay. Briefly, the procedure used for in vitro transcription andtranslation was as described in Short Protocols In Molecular Biology,Third Edition, Ed. Ausubel et al., Wiley & Sons Pub., (1995), which isherein incorporated by reference. About 20 micrograms of plasmid DNA wasdigested to completion with HindIII restriction endonuclease in a 100microliter reaction. 20 microliters of the plasmid digest, or about 4micrograms of linearized plasmid DNA, was used in an in vitrotranscription reaction to generate mRNA for producing PhnO or CTP-PhnOprotein product in later translation reactions. Transcription reactionsconsisted of 20 microliters of linearized plasmid DNA, 20 microliters ofa 5×transcription buffer (200 mM TrisHCl pH 8.0, 40 mM MgCl₂, 10 mMspermidine and 250 mM NaCl), 20 microliters of 5×ribonucleosidetriphosphate mix (5 mm each ATP, CTP, UTP, 5 mM diguanosine triphosphate(G-5′ppp5′-G)TP, 5 mM GTP), 10 microliters 0.1 M dithiothreitol (DTT),10 microliters RNasin™ (a pancreatic ribonuclease inhibitor mixture fromPromega), 4 microliters RNA polymerase (T7 or T3, New England Biolabs,Inc.), and distilled, deionized water to 100 microliters. Each reactionwas incubated at 37° C. for one hour. 4.5 microliters of each reactionwas analyzed on a 1.4% agarose formaldehyde gel to ensure that eachreaction produced adequate RNA template for the following translationstep.

[0253] 20 microliters of the transcription reactions were used forproducing [35S]-methionine labeled PhnO proteins for use in achloroplast import assay. Briefly, RNA was mixed with 6 microliters ofan aqueous amino acid mixture without methionine, 15 microliters of[³⁵S]-methionine (1400 Ci/mmol, Amersham), and 200 microliters of arabbit reticulocyte lysate. These reactions were incubated at 37° C. fortwo hours and placed on dry ice for storage. A 10 microliter sample ofeach reaction was analyzed on a 15% SDS-PAGE gel. Gels were vacuum driedand placed directly onto the emulsion side of KODAK™ X-O-MAT™ film forautoradiography. The results indicated that each plasmid producedrespective peptides of predicted molecular mass for PhnO (pMON15035) andCTP-PhnO (pMON15036) in sufficient quantity to test for uptake intochloroplasts in an import assay.

[0254] Intact chloroplasts were isolated from one head of deveinedRomaine lettuce according to Edelman et al., Methods in ChloroplastMolecular Biology, Elsevier Biomedical Press, Chap. 86, 1982. One literof grinding buffer (GR-buffer) stock was prepared (2 mM NaEDTA, 1 mMMgCl2, 1 mM MnCl2, 50 mM Hepes-KOH pH 7.5, and 0.33 mM sorbitol).Immediately before use, 890 mg of ascorbic acid was added to 900 ml ofGR-buffer stock solution. One head of torn, deveined Romaine lettuce wasmixed with 900 ml GR-buffer and emacerated by mixing in a Waring blenderthree times for three seconds each time at high speed. The slurry wasfiltered through four layers of Miracloth, and the filtrate wascentrifuged at 5,000 RPM for 10 minutes at 4° C. in a SORVALL™ GS-3rotor. The supernatant was decanted and the pellet resuspended with aglass rod in 4 milliliters of GR-buffer. Chloroplasts were isolated bycentrifugation through a Percoll gradient. 80% Percoll was prepared bymixing 16 mls of PBF-Percoll with 4 mls of 5×Buffer (10 mM EDTA, 5 mMMgCl₂, 5 mM MnCl₂, 250 mM Hepes-KOH, 30 grams sorbitol, 490 mgNaAscorbate, 85.5 mg glutathione to 100 mls with ddH₂O). A 40% Percollsolution was prepared by combining 8 mls PBF-Percoll with 4 mls 5×Bufferand 8 mls of ddH₂O. A Percoll gradient was prepared in a 30 ml Corextube by layering 10 mls of 40% Percoll onto 10 mls of 80% Percoll.Chloroplasts were isolated by layering the resuspended chloroplasts ontothe percoll gradient, spinning at 9,500 RPM for ten minutes in an SS-34SORVALL™ swinging bucket rotor at 4° C. for ten minutes with the brakeon. Broken chloroplasts remain in the upper layer and were pipetted off.The intact chloroplasts were located at the interface of the 40/80%Percoll gradient and were removed to a new 30 ml COREX™ tube. Theisolated chloroplasts were washed two times with GR-buffer andcentrifuged for collection after each wash in a SS-34 rotor at 6,000 RPMfor ten minutes at 4° C. with the brake off. Isolated, washedchloroplasts were resuspended in 1 ml sterile 50 mM Hepes-KOH pH 7.7,330 mM sorbitol by gently stirring with a glass rod, and the chlorophyllconcentration of the slurry was determined. 5 mls of an 80% acetonesolution was added to 20 microliters of the chloroplast slurry andvortexed gently. The resulting mixture was filtered through a Whatman™#1 filter paper into a culture tube. The absorbance of the filtrate wasdetermined at 645 nm and 663 nm against an 80% acetone blank. Thechlorophyll concentration in micrograms per ml was determined accordingto equation #1 as [chlorophyll μg/ml]=[A₆₄₅+[A₆₆₃*(8.02)]. The mass ofthe chlorophyll in μg is calculated by taking the amount of chlorophyllmeasured in μg/ml and multiplying by the volume into which thechloroplasts were resuspended (equation #2), which is 5 mls in thisexample. Thus, the concentration of chlorophyll in μg/μl in the measuredsample is equivalent to the value determined in equation #2 divided bythe volume of the sample measured, which in this example is 20 μl . Inthis example, A₆₄₅ was determined to be 0.496, and A₆₆₃ was determinedto be 1.0814. Thus, the concentration of chlorophyll in the measuredsample was 4.67 μg/μl. The concentration of chlorophyll in thechloroplast slurry was adjusted to 4.0 μg/μl with Hepes-KOH pH 7.7, 330mM sorbitol solution and the resulting chloroplast suspension was storedon ice in the dark.

[0255] A typical 300 microliter uptake experiment contained 5 mM ATP,8.3 mM unlabeled methionine, 322 mM sorbitol, 58.3 mM Hepes-KOH (pH8.0), 50 microliters reticulocyte lysate translation products, andintact chloroplasts (about 200 microgram chlorophyll). The uptakemixtures were gently rocked at room temperature in 10×75 mm glass tubes,directly in front of a fiber optic illuminator set at maximum lightintensity using a 150 Watt bulb. Two separate 70 microliter samples ofeach uptake mix were removed at 0, 5, 10 and 15 minutes. One sample wascentrifuged over 100 microliter silicone-oil gradients in 150 microliterpolyethylene tubes by centrifugation at 11,000×g for 30 seconds, andimmediately frozen in dry ice. Under these conditions, the intactchloroplasts form a pellet under the silicone-oil layer and theincubation medium containing the reticulocyte lysate remains floating onthe surface of the interface. The other sample was treated with protease(one tenth volume or 7 microliters of 0.25 mg/ml each trypsin andchymotrypsin protease mixture) for thirty minutes on ice, then subjectedto silicone-oil separation and frozen on dry ice. The chloroplastpellets were then resuspended in 50-100 microliters of a lysis buffer(10 mM Hepes-KOH pH 7.5, 1 mM PMSF, 1 mM benzamidine, 5 mMε-amino-n-caproic acid, and 30 micrograms per ml aprotinin) andcentrifuged at 15,000×g for 20 minutes to pellet the thylakoidmembranes. The cleared supernatant (stromal proteins) from this spin,and an aliquot of the reticulocyte lysate incubation medium from eachuptake experiment, were mixed with an equal volume of 2×SDS-PAGE samplebuffer and analyzed on a 15% SDS-PAGE gel, dried, and exposed to film asdescribed above. Chloroplasts exposed to [³⁵S]-methionine labeledCTP-PhnO contained [³⁵S]-labeled protein of a size consistent with thepredicted CTP-processed form of PhnO, while chloroplasts exposed tomethionine labeled PhnO were devoid of labeled protein. Labeled proteinimported into the chloroplasts was also protease resistant. Theseresults indicated that PhnO could be targeted to chloroplasts when fusedto a plastid targeting peptide sequence.

Example 8

[0256] This example illustrates the identification and characterizationof plants transformed with an AMPA acyltransferase.

[0257] A wide variety of plant species have been successfullytransformed using any number of plant transformation methodologies wellknown in the art. In particular, Agrobacterium tumefaciens mediatedplant transformation is the preferred method presently in use, however,ballistic methods which increase delivery of naked DNA directly to plantcells through microprojectile bombardment are also very effective inproducing recombinantly transformed plants. In addition, methods whichinvolve the use of liposomes, electroporation, chemicals that increasefree DNA uptake, and transformation using viruses or pollen arealternatives which can be used to insert DNA constructs of thisinvention into plant cells. Plants which can be transformed by thepractice of the present invention include but are not limited to corn,wheat, cotton, rice, soybean, sugarbeet, canola, flax, barley, oilseedrape, sunflower, potato, tobacco, tomato, alfalfa, lettuce, apple,poplar, pine, eucalyptus, acacia, poplar, sweetgum, radiata pine,loblolly pine, spruce, teak, alfalfa, clovers and other forage crops,turf grasses, oilpalm, sugarcane, banana, coffee, tea, cacao, apples,walnuts, almonds, grapes, peanuts, pulses, petunia, marigolds, vinca,begonias, geraniums, pansy, impatiens, oats, sorghum, and millet. DNAmolecules for use in the present invention can be native or naturallyoccurring genes or chimeric genes constructed from useful polynucleotidesequences including promoters, enhancers, translated or non-translatedleaders, sequences encoding signal peptides, sequences encoding transitpeptides, structural genes, fusions of structural genes, terminators,introns, inverted repeats or direct repeats, linkers, andpolyadenylation sequences. DNA sequences contemplated in this inventioninclude single and double stranded polynucleotide sequences, linearsequences, and covalently closed circular polynucleotide sequences,plasmids, bacmids, cosmids, bacterial artificial chromosomes (BAC's),yeast artificial chromosomes (YAC's), and viral DNA and RNA sequences.In consideration of Agrobacterium mediated plant transformation,suitable plant transformation vectors include those derived from a Tiplasmid of Agrobacterium tumefaciens, as well as those disclosed, forexample by Herrera-Estrella (1983), Bevan (1984), Klee (1985) and EPOpublication 120,516 (Schilperoort et al.). In addition to planttransformation vectors derived from the Ti or root-inducing (Ri)plasmids of Agrobacterium, alternative methods as described above can beused to insert the DNA constructs of this invention into plant cells.

[0258] Plasmids used for plant transformation generally were constructedfrom vectors which have been described elsewhere, particularly in U.S.Pat. No. 5,463,175 (Barry et al., 1995), which is herein incorporated byreference. Plasmids were constructed and maintained in E. coli using Tn7aminoglycoside adenylyltransferase resistance (aad gene, commonlyreferred to as streptomycin/spectinomycin or Spc/Str resistance), whichis also a determinant for selection and maintenance in Agrobacterium.Other plasmid maintenance and selectable markers well known in the artfor use in E. coli were also used, consisting essentially of neomycinphosphotransferase, gentamycin acetyltransferase, and beta lactamasegenes alone or present in combination on a single replicon or vector.Plasmids generally contain oriV, a replication origin derived from thebroad host range plasmid RK2, and ori322 and bom (origin of replicationfor maintenance in E. coli, and basis of mobility for conjugationaltransfer respectively) sequences derived from plasmid pBR322.

[0259] A phnO gene encoding an AMPA acyltransferase was inserted intoexpression cassettes in plant transformation vectors. These cassettesgenerally contain the following elements in sequential 5′ to 3′ order: asequence comprising a plant operable promoter, a sequence encoding achloroplast or plastid transit peptide, a cloning site or sitescontained within a polylinker, and a plant functional 3′ nontranslatedregion. Expression cassettes often are constructed to contain uniquerestriction sites flanking the cassette domain so that the entirecassette can be excised from one plasmid and placed into other similarlyconstructed plasmid vectors. Restriction sites comprised of eight basepair recognition sequences are preferred, and most cassettes in thepresent invention are flanked at least on one end by a NotI restrictionendonuclease recognition site. Preferred promoters are the figwortmosaic virus promoter, P-FMV (Gowda et al., 1989), the cauliflowermosaic virus 35S promoter CaMV 35S (Odell et al., 1985), or the enhancedCaMV 35S promoter (U.S. Pat. No. 5,196,525; Kay et al., 1987). A numberof other promoters which are active in plant cells have been describedin the literature. Such promoters may be obtained from plants or plantviruses and include, but are not limited to the nopaline synthase (NOS)and octopine synthase (OCS) promoters which are carried ontumor-inducing plasmids generally found within virulent and non-virulentstrains of Agrobacterium tumefaciens, the cauliflower mosaic virus(CaMV) 19S promoter, the comalina yellow mottle virus promoter, thesugar cane bacilliform DNA virus promoter, the peanut chlorotic streakvirus promoter, the rice actin promoter, and the light-inducibleribulose 1,5-bisphosphate carboxylase small subunit promoter(ssRUBISCO). These promoters can used to create various types of DNAconstructs useful for gene expression in plants (see for example Barryet al. U.S. Pat. No. 5,463,175). Particularly desirable promoters whichare contemplated because of their constitutive nature are theCauliflower Mosaic Virus 35S (CaMV35S) and the Figwort Mosaic Virus 35S(FMV35S) promoters which have previously been shown to produce highlevels of expression in most plant organs. Other promoters which woulddirect tissue specific or targeted expression are also contemplated, forexample in tissue such as leaves, meristem, flower, fruit and organs ofreproductive character. In addition, chimeric promoters are alsoenvisioned. Nopaline synthase gene (NOS 3′) and the pea ribulosebisphosphate carboxylase synthase E9 gene (E9 3′) 3′ nontranslatedtermination and polyadenylation sequences were also used.

[0260] Expression cassettes consisting of a AMPA acyltransferasestructural gene inserted downstream of a promoter and between a sequenceencoding a chloroplast targeting peptide and a 3′ nontranslated sequencewere generally present on a plant transformation vector. Expressioncassettes were generally flanked on either end of the cassette by anopaline type T-DNA right border region on one end and a left borderregion on the other end, both border regions derived from pTiT37 (Fraleyet al., 1985). Some plant transformation vectors only contained theright border region, required for initiation of T-DNA transfer fromAgrobacterium to the host cell. Most plant transformation vectors alsocontained a GOX (glyphosate oxidoreductase) gene, as described above,and in U.S. Pat. No. 5,463,175. GOX enzyme expressed from these vectorswas generally targeted to the chloroplast when inserted into the plantgenome.

[0261] Plant transformation vectors were mobilized into the ABIAgrobacterium strain A208 carrying the disarmed Ti plasmid pTiC58(pMP90RK)(Koncz and Schell, 1986). The Ti plasmid does not carry theT-DNA phytohormone genes which induce crown gall formation. Mating ofthe plant vector into ABI was done by the triparental conjugation systemusing the helper plasmid pRK2013 (Ditta et al., 1980). Alternatively,the plant transformation plasmid can be introduced into the ABI strainby electroporation as described by Mattanovich et al. (Efficienttransformation of Agrobacterium spp. by electroporation., Nucleic AcidsRes. (1989), 17(16), 6747), which is herein incorporated by reference.When plant tissue is incubated with the ABI::plant vector conjugate, therecombinant vector is transferred to the plant cells by the virfunctions encoded by the disarmed pTiC58 plasmid. Ideally, therecombinant vector opens at the T-DNA right border region, and the DNAbetween the right and left border sequences is transferred directionallyand inserted into the host plant genome, although the entire recombinantplant transformation vector sequence may be transferred and inserted.The pTiC58 Ti plasmid does not transfer to the plant cells but remainsin the Agrobacterium donor.

[0262] Recombinant plants can be regenerated from plant cells or planttissue which has been transformed with a functional AMPA acyltransferasestructural gene. The choice of methodology for the regeneration step isnot critical, with suitable protocols being available for hosts fromLeguminosae (alfalfa, soybean, clover, etc.), Umbelliferae (carrot,celery, parsnip), Cruciferae (cabbage, radish, rapeseed, etc.),Cucurbitaceae (melons and cucumber), Gramineae (wheat, rice, corn,etc.), Solanaceae (potato, tobacco, tomato, peppers), and various floralcrops. See for example, Ammirato, 1984; Shimamoto, 1989; Fromm, 1990;and Vasil, 1990). Recombinant plants which have been transformed with anAMPA acyltransferase can also be selected on medium containing AMPA. Theappropriate inhibitory concentration of AMPA can readily be determinedby one of ordinary skill in the art for any particular host by screeningfor AMPA toxicity as described in example 1. Alternatively, when AMPAacyltransferase is transformed into plants previously transformed withGOX and selected for growth on glyphosate, either AMPA or glyphosate canbe used as the selective ingredient for selecting for transformationevents which express sufficient levels of AMPA acyltransferase enzyme.Glyphosate must be applied at levels which would otherwise be inhibitoryto a recombinant plant expressing GOX and selected for growth onglyphosate, due to the increased level of AMPA which may be produced asa result of GOX mediated glyphosate degradation. In plants which expressrecombinant GOX enzyme, exposure to increasing levels of glyphosate hasbeen shown to induce yellowing or chlorosis of the leaves, stuntedgrowth characteristics, and infertility. AMPA acyltransferase expressedcoordinately or in combination with GOX expression can overcome thesedetrimental effects. It is also possible to use AMPA as a planttransformation selectable marker as an alternative to glyphosateselection.

[0263] Tobacco

[0264] Tobacco plants were transformed with a phnO gene. A tobacco leafdisc transformation procedure employed healthy tissue from a leaf ofabout one month old. After a 15-20 minute surface sterilization with 10%CLOROX™ plus a surfactant, leaves were rinsed three times in sterilewater. Leaf discs were punched with a sterile paper punch, and placedupside down on MS 104 media (4.3 g/l MS salts, 30 g/l sucrose, 2 ml/l500× B5 vitamins, 0.1 mg/l NAA, and 1.0 mg/l BA), and pre-cultured forone day. Discs were then inoculated with an 1:5 diluted overnightculture of disarmed Agrobacterium ABI containing the subject vector(final culture density about 0.6 OD as determined at 550 nm). Theinoculation was done by placing the discs in sterile centrifuge tubesalong with the culture. After thirty to sixty seconds, the liquid wasdrained off and the discs were blotted between sterile filter paper. Thediscs were then placed upside down on a filter disc on MS 104 feederplates and incubated for 2-3 days. After this co-culture period, thediscs were transferred, still upside down, to selection platescontaining MS 104 media. After 2-3 weeks, callus formed, and individualclumps were separated from the leaf discs. Shoots were cleanly cut fromthe callus when they were large enough to distinguish from stems. Theshoots were placed on hormone-free rooting media (MSO: 4.3 g/l MS salts,30 g/l sucrose, and 2 ml/l 500×B5 vitamins) with selection. Roots formedin 1-2 weeks. Any leaf callus assays are preferably done on rootedshoots while still sterile. Rooted shoots were placed in soil and weremaintained in a high humidity environment (ie: plastic containers orbags). The shoots were hardened off by gradually exposing them toambient humidity conditions.

[0265] Three tobacco transformation events, designated as lines 33476,36779, and 37235 were selected for further analysis. pMON17226 (Barry etal., U.S. Pat. No. 5,463,175, 1995) was used to produce plant line 33476which contains an FMV-CTP-GOX gene construct. Lines 36779 and 37235 wereproduced using pMON17261, which is a plasmid derived from pMON17226which contains NotI cassette containing an FMV-CTP-PhnO gene sequence(SEQ ID NO: 11) in addition to FMV-CTP-GOX. The NotI cassette wasconstructed as follows. The sequence encoding CTP, represented by SEQ IDNO: 9, was excised from pMON17058 as a BglII to NcoI fragment andinserted into pMON15028, forming a sequence represented by SEQ ID NO: 11in which the CTP coding sequence was upstream of and in frame with thePhnO coding sequence represented within SEQ ID NO: 7. The resultingconstruct was designated as pMON15029. The CTP-PhnO coding sequence wasexcised from pMON15029 on a BglII to SacI fragment and combined withpMON17063 fragments to produce pMON15038 pMON17063 was disassembledusing restriction digestion to provide parts necessary for pMON15038construction. pMON17063 was digested with SacI and HindIII to produce avector backbone into which a promoter fragment and the CTP-PhnO sequencewere inserted. pMON17063 was also digested in a separate reaction withHindIII and BglII to produce a fragment containing an FMV promotersequence. The promoter fragment and the CTP-PhnO fragment were ligatedtogether in a reaction along with the vector backbone fragment toproduce pMON15038, containing a NotI cassette harboring a sequenceencoding a chloroplast targeted PhnO peptide expressed from an FMVpromoter and flanked downstream by a NOS E9 3′ transcription terminationand polyadenylation sequence. This NotI sequence was excised frompMON15038 and inserted into the unique NotI site in pMON 17241 toproduce pMON17261, containing a chloroplast targeted GOX coding sequenceexpressed from an FMV promoter and flanked downstream by an E9 3′sequence, along with the CTP-PhnO coding sequence and expressioncassette. Transformation events derived from this vector are expectednot only to be resistant to glyphosate, but to provide resistance toAMPA phytotoxicity as well. Lines 36779 and 37235 derived from pMON17261were analyzed for the presence of genes encoding glyphosateoxidoreductase and AMPA acyltransferase by PCR, for the presence of GOXand PhnO enzymes by western blot, and for the presence of metabolitesproduced as a result of GOX mediated [¹⁴C]-glyphosate degradation byHPLC.

[0266] Line 33476, obtained as a transformation event derived frompMON17226, was selected as a “GOX only” control. Lines 36779 and 37235demonstrated different phenotypes upon exposure to glyphosate and wereselected as glyphosate resistant events arising after transformationwith pMON17261. Line 37235 became bleached or yellowed upon exposure toglyphosate, similar in phenotype to the GOX only line 33476. However,line 36779 displayed no such bleaching effect. DNA was extracted fromleaf tissue for each of these events as well as from wt Samsun tobaccoleaf, and subjected to PCR to determine the presence or absence of thetransforming phnO gene.

[0267] Genomic DNA isolated from transformed tobacco lines was used asthe template DNA in a PCR reaction and reaction products were comparedto wild type Samsum tobacco. PCR reactions consisted of 50 microliterstotal volume containing 10×amplification buffer, 1.5 mM MgCl₂,deoxynucleotide mix with each at 1 mM, 50-100 ng genomic DNA, primerseach at a final concentration of 16.8 pM, and 1.5 units of AmpliTaq DNApolymerase (Cetus/Perkin Elmer). Primers (synthesized to order byGENOSYS) consisted of the sequences as set forth in SEQ ID NO: 21 andSEQ ID NO: 22. SEQ ID NO: 21 is a 20 base pair sequence capable ofpriming the synthesis of the P2A phnO gene sequence (SEQ ID NO: 7) andhybridizes to the first twenty nucleotides of the coding sequence inthat gene. SEQ ID NO: 22 is also a 20 base pair sequence, but is capableof priming synthesis of a phnO gene from the terminal coding sequenceinto the structural coding region and hybridizes to the terminal twentynucleotides of the sequence encoding PhnO. Amplification conditionsconsisted of three cycles of 97° C. for one minute, 60° C. for twominutes, and 72° C. for two minutes, followed by 37 cycles of 94° C. forone minute, 60° C. for two minutes, and 72° C. for two minutes, followedgenerally by a 4° C. soak. 10 microliter samples were generally analyzedby 1% TAE agarose gel electrophoresis to resolve the relevant bands fromresidual primers. Upon ethidium bromide staining of the product gels, aphnO gene amplification product about 432 base pairs as judged by themigration position versus HindIII digested lambda molecular weightmarkers appeared only in the line 33779 extracts, indicating thepresence of the phnO gene in that line.

[0268] Seed from Ro transformation events were obtained after selfcrossing in growth chamber conditions. Ro seed were cured and planted togenerate R1 progeny. Source leaves of R1 progeny at the five leaf stagewere exposed to [¹⁴C]-glyphosate by spotting a 2 microliter sample ontoeach vein (50 microliters of [¹⁴C]-glyphosate Na+ salt, 517,000dpm/microgram, 0.42 microgram/microliter mixed with 10 microliters ofglycerol). Each leaf received several spots depending on the number ofveins on that leaf. Three days later 15 additional 2 microliter spotswere applied to each leaf. Two weeks later, five 2 microliter spots wereapplied to each of two leaves on each plant. These were new leaves andwere not the older leaves to which glyphosate was initially applied.Five days after this last application, about 300 milligrams of tissuewas sampled from two sink leaves on each plant. The samples from eachplant were homogenized in separate 1 ml volumes of deionized water,centrifuged at 9,000 RPM in a microcentrifuge, and the aqueous volumeswere collected and stored on ice. Extracts were analyzed by HPLC for thepresence of [¹⁴C] labeled metabolites as in Example 2. The extractobtained from line 33476 (GOX) contained only [¹⁴C]-AMPA. The extractobtained from line 37235 contained non-metabolized [¹⁴C]-glyphosate aswell as a trace but measurable amount of [¹⁴C]-AMPA. OnlyN-acetyl-[¹⁴C]-AMPA was observed in the extract obtained from line36779. These results are consistent with the PCR data which indicatedthat line 36779 contained at least one copy of the phnO gene. Inaddition, the lack of a bleaching effect in line 36779 after exposure toglyphosate is consistent with the presence of functional GOX and PhnOenzymes and the absence of detectable [¹⁴C]-AMPA.

[0269] Cotton

[0270] A recombinant phnO gene was transformed into Coker 312 varietycotton (Gossypium hirsutum L.). Glyphosate tolerant cotton lines wereproduced by Agrobacterium mediated plant transformation using doubleborder binary plasmid vectors containing either (1) gox, anAchromobacter sp. strain LBAA gene encoding a glyphosate-metabolizingenzyme glyphosate oxidoreductase (GOX), (2) the gox gene and an E. coliphnO gene encoding PhnO, or (3) the gox/phnO double gene construct alongwith an Agrobacterium strain CP4 gene encoding5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). All vectors arecapable of replication in both Agrobacterium tumefaciens and E. colihosts, and contain an aminoglycoside adenylyltransferase gene (aad)conferring resistance to aminoglycoside such as spectinomycin orstreptomycin and providing a method for plasmid maintenance.

[0271] pMON17241 contains a recombinant gene consisting of a 35S FMVpromoter linked 5′ to an Arabidopsis thaliana ribulose-1,5-bisphosphatecarboxylase small subunit (SSU1A) gene sequence encoding a plastid orchloroplast targeting peptide (Timko et al., 1988) which istranslationally fused to a gox gene coding sequence, which is linked 3′to a 3′ untranslated region, designated E9, from a pearibulose-1,5-bisphosphate carboxylase gene.

[0272] pMON17213 is a double gene plant transformation vector containingexpression cassettes comprising (1) a 35S FMV promoter linked to asequence encoding an Arabidopsis thaliana EPSPS chloroplast targetingpeptide linked in-frame to a strain CP4 EPSPS coding sequence, which islinked 3′ to an E9 3′ untranslated region; and (2) a 35S FMV promoterlinked to an SSU1A gene sequence encoding a plastid targeting peptidelinked in-frame to a GOX coding sequence, which is linked 3′ to a NOS 3′termination sequence.

[0273] pMON17261, described above, is a double gene plant transformationvector containing expression cassettes comprising (1) an FMV 35Spromoter linked to an SSU1A chloroplast targeting peptide codingsequence linked in-frame to a GOX coding sequence, which is flankeddownstream by the E9 3′ untranslated region; and (2) an FMV 35S promoterlinked to an SSU1A chloroplast targeting peptide coding sequence (SEQ IDNO: 9) linked in-frame to a PhnO coding sequence (SEQ ID NO: 7), whichis linked 3′ to a NOS 3′ sequence.

[0274] pMON10151 is a double gene plant transformation vector containingexpression cassettes comprising (1) an FMV 35S promoter linked to anSSU1A chloroplast targeting peptide coding sequence (SEQ ID NO: 9)linked in-frame to a PhnO coding sequence (SEQ ID NO: 7), which isflanked downstream by a NOS 3′ sequence; and (2) an enhanced 35Spromoter linked to an SSU1A chloroplast targeting peptide codingsequence linked in-frame to a GOX coding sequence which is flankeddownstream by a NOS 3′ sequence.

[0275] pMON10149 is a triple gene plant transformation vector containingexpression cassettes comprising (1) an FMV 35S promoter and a petuniaHSP70 5′ untranslated leader sequence linked to an SSU1A chloroplasttargeting peptide coding sequence linked in-frame to an EPSPS codingsequence, which is flanked downstream by the E9 3′ termination andpolyadenylation sequence; (2) an FMV 35S promoter linked to an SSU1Achloroplast targeting peptide coding sequence (SEQ ID NO: 9) linkedin-frame to a PhnO coding sequence (SEQ ID NO: 7), which is flankeddownstream by a NOS 3′ sequence; and (3) an enhanced 35S CaMV promoterlinked to an SSU1A chloroplast targeting peptide coding sequence linkedin-frame to a GOX coding sequence, which is flanked downstream by anopaline synthase 3′ polyadenylation sequence (NOS 3′).

[0276] Plasmid vectors were assembled in E. coli K12 strains and matedinto a disarmed ABI Agrobacterium strain. Aminoglycoside resistantAgrobacterium strains were used to transform Coker 312 derived hypocotylsections with modifications as described by Umbeck et al. (1987) andUmbeck (U.S. Pat. No. 5,159,135 (1992), incorporated herein byreference), except that plants were regenerated with modificationsdescribed by Trolinder and Goodin (1987). Selection for glyphosateresistance produced several lines of cotton callus, which weresubsequently determined by PCR of genomic DNA to contain the respectivegenes encoding EPSPS, GOX or PhnO transferred from Agrobacterium.Additionally, these same callus lines were determined by Western blotanalysis to express the desired genes. After plant regeneration, wholecotton plants which contained the indicated coding sequences wererecovered.

[0277] Previously identified plants transformed with a double geneglyphosate resistance cassette comprised of EPSPS and GOX encoding geneswere determined to be resistant to glyphosate when applied at 48 ouncesper acre through the 6-7 leaf stage, however severe bleaching of theleaves was observed. This phytotoxic effect was presumed to be due tothe formation of AMPA as a result of GOX mediated glyphosatedegradation. To test this, AMPA was sprayed at three different ratesonto wild type Coker 312 plants. Leaf chlorosis and stunted growth wasobserved in plants at four days post-application of glyphosate at 640ounces per acre and at eight days post-application of 64 ounces peracre. These results suggested that the phytotoxic effect observed inEPSPS/GOX transformed cotton plant lines was a result of GOX mediatedAMPA production in plants, and that the phytotoxic effect may beobviated by co-expression of an AMPA acyltransferase along with GOX. Totest this, cotton plants expressing GOX or GOX plus EPSPS alone or incombination with PhnO expression were treated with [¹⁴C]-glyphosate, andthe metabolism of the isotope labeled glyphosate was monitored in leaftissue seven days after application.

[0278] Coker 312 glyphosate resistant recombinant cotton line 4416 wasselected as a glyphosate resistant cotton line after transformation withpMON10149, a triple gene Agrobacterium tumefaciens mediated doubleborder plant transformation vector containing chloroplast targetedEPSPS, GOX, and PhnO, each expressed independently from separate 35Spromoters. Several 4416 R3 plants were raised from R2 seed. One leaf ofeach plantlet at the three or four stage was treated with a mixture ofROUNDUP ULTRA™ commercial herbicide mixture (Lot No. GLP-9701-7428-F)which had been fortified with [¹⁴C]-glyphosate (Code No. C-2251). TheROUNDUP ULTRA™ was shown to be 30.25% glyphosate acid by weight and the[¹⁴C]-glyphosate had a radiochemical purity of 97.3% and a specificactivity of 36.36 mCi/mmol. The treatment solution consisted ofapproximately 38 μL containing 1.60×10⁶ dpm with a [¹⁴C]-glyphosatespecific activity of 1.713×10³ dpm/μg glyphosate acid. Three or sevendays after topical application the treated leaves were rinsed withwater, frozen in liquid nitrogen, fractured with a spatula and thenground using a TEKMAR™ tissuemizer in 10 mL of water. The leaf extractswere adjusted to pH 3.5-4.0 with 1N HCl and approximately 4-8000 dpmwere analyzed for the presence of [¹⁴C]-metabolites by HPLC with liquidscintillation vial collection and detection (HPLC/LSC) as described inexample 2. The new growth including the meristem and new leaves thatemerged following topical application were also extracted and analyzedfor [¹⁴C]-metabolites. The results are shown in Table 13. TABLE 13[¹⁴C]-Glyphosate Metabolism In Glyphosate Resistant Cotton % [¹⁴C]metabolite in Glyphosate % [¹⁴C] metabolite in Treated Leaf Extract. ..* New Growth Extract . . .* Line 4416 N-Acetyl- N-Acetyl Plant #Glyphosate AMPA AMPA Glyphosate AMPA AMPA MD03 55.2 2.5 37.4 nd** nd93.4 MD04 94.6 2.1  1.7 97.9  nd nd A01 48.6 2.1 44.7 0.9 0.2 95.8 A0267.3 2.0 29.1 0.7 0.2 96.5 A03 48.8 2.0 43.4 1.2 nd 94.0 A04 19.4 1.673.9 1.5 nd 94.0 A05 59.9 2.2 31.1 2.2 0.2 95.2 A06 38.2 nd 60.9 1.5 0.293.5 A07 64.1 nd 26.8 1.4 0.5 93.9 A08 90.9 2.0  1.9 91.2  2.5  1.9

[0279] Analysis of the water rinsed glyphosate treated leaves indicatedthe presence of significant levels of N-acetyl-[¹⁴C]-AMPA in eight ofthe ten plants tested. These levels represented 27-74% of the isotopeextracted from the treated leaves. The remaining activity was almostentirely [¹⁴C]-glyphosate. Very little of the [¹⁴C] isotope was presentas [¹⁴C]-AMPA. The remaining two plants had very limited ability tometabolize glyphosate as indicated by the high levels of[¹⁴C]-glyphosate remaining on or in the leaves. One of these plants alsoshowed signs of stunting seven days after treatment, indicatingglyphosate phytotoxicity.

[0280] Analysis of new growth in the ten plants tested showed that thepredominant form of [¹⁴C] labeled metabolite present wasN-acetyl-[¹⁴C]-AMPA at greater than 90% of the total radioisotope in thesamples. In contrast, more than 90% of the isotope in the remaining twoplants was in the form of [¹⁴C]-glyphosate, consistent with the analysisof the extract from the treatment leaf for these two plants.

[0281] The metabolism of [¹⁴C]-glyphosate in recombinant cotton lines4268 (GOX/PhnO) and 3753 (EPSPS/GOX) was also studied. Plants in thisstudy were treated as indicated above for cotton line 4416, by applyingdroplets of ROUNDUP ULTRA fortified with [¹⁴C]-glyphosate to a singleleaf on each plant at the three to four leaf stage. Treated leaves wereharvested and rinsed with water, then ground and extracted, and extractswere analyzed by HPLC as described above for the presence of[¹⁴C]-glyphosate, [¹⁴C]-AMPA, and N-acetyl-[¹⁴C]-AMPA. New growth,including the meristem and new leaves that emerged following applicationwere also extracted and analyzed. The results are shown in Table 14.TABLE 14 [¹⁴C]-Glyphosate Metabolism In Glyphosate Resistant Cotton *%[¹⁴C] metabolite in Glyphosate *% [¹⁴C] metabolite in Treated LeafExtract. . . New Growth Extract. . . Plant Glyphosate AMPA N-Acetyl-AMPAGlyphosate AMPA N-Acetyl-AMPA GOX/PhnO Plants B01 76.7 3.0 14.0 3.4 1.089.9 B02 63.9 4.8 25.0 1.1 1.5 91.5 B03 54.4 3.2 36.4 0.8 nd 94.7 B0458.3 5.7 28.9 1.1 1.2 91.0 EPSPS/GOX Plants C01 59.8 26.6  nd  3.7285.7  nd C02 92.7 2.1  0.8 92.8  0.8 nd C03 81.2 10.7  nd 13.5  72.0  1.9 C04 86.2 6.4  1.0 13.9  76.2  nd

[0282] Significant levels of N-acetyl-[¹⁴C]-AMPA were present in thetreated leaves of all four line 4268 plants (GOX/PhnO; B01-B04). Incontrast, N-acetyl-[¹⁴C]-AMPA was not detectable in extracts obtainedfrom line 3753 plants (EPSPS/GOX; C01-C04). Three of these plantscontained significant levels of [¹⁴C]-AMPA in treated leaf extracts,ranging from 6-27%. One line 3753 plant was deficient in the conversionof [¹⁴C]-glyphosate to N-acetyl-[¹⁴C]-AMPA, and this plant also appearedto be stunted.

[0283] 90-95% of the [¹⁴C] isotope in extracts of new growth from line4268 plants was determined to be in the form of N-acetyl-[¹⁴C]-AMPA.However, 72-86% of the [¹⁴C] isotope in extracts of new growth fromthree of the line 3753 plants was determined to be [¹⁴C]-AMPA, with[¹⁴C]-glyphosate accounting for the remainder of the isotope in thesetissues. 93% of the isotope obtained from line 3753 plant number C02 wasdetermined to be [¹⁴C]-glyphosate, consistent with the lack ofglyphosate metabolism in the application leaf as well as the observedstunting. In addition, growth regions of all line 3753 plants werediscolored and yellow following treatment, but improved with time. Byharvest, new growth leaves became mottled.

[0284] These results are consistent with the presence of active gox andphnO gene products in the indicated plants. The GOX and PhnO proteinsare metabolizing glyphosate to AMPA and N-acetyl-AMPA in the predictedmanner, and line 4268 plant extracts provide a similar metabolic patternto that observed with line 4416 plant extracts as judged by HPLC and byphenotypic observation. In both lines, the predominant [¹⁴C] product innew growth tissue extracts after [¹⁴C]-glyphosate application isN-acetyl-[¹⁴C]-AMPA. The phytotoxicity as observed by discoloration ofplant leaves in line 3753 after glyphosate application is associatedwith the lack of an AMPA N-acyltransferase activity. In contrast, thepresence of an AMPA N-acyltransferase activity in both the 4416 and the4268 plant lines resulted in a lack of phytotoxic effects observed inline 3753 plants.

[0285] Canola

[0286] Canola plants were transformed with the vectors pMON17138 andpMON17261 and a number of plant lines of the transformed canola wereobtained which exhibited glyphosate tolerance. Plants were transformedaccording to the method described in Barry et al. (U.S. Pat. No.5,633,435). Briefly, Brassica napus cv Westar plants were grown incontrolled growth chamber conditions as described. Four terminalinternodes from plants just prior to bolting or plants in the process ofbolting but before flowering were removed and surface sterilized in 70%v/v ethanol for one minute, then in 2% w/v sodium hypochlorite fortwenty minutes, then rinsed three times with sterile distilled deionizedwater. Stems with leaves attached could be refrigerated in moist plasticbags for up to three days prior to sterilization. Six to seven stemsegments were cut into 5 mm discs with a Redco Vegetable Slicer 200maintaining orientation of basal end. Stem discs (explants) wereinoculated with 1 milliliter of ABI Agrobacterium tumefaciens strainA208 containing a recombinant plant transformation plasmid prepared asdescribed above. Explants were placed basal side down in petri platescontaining 0.1×standard MS salts, B5 vitamins, 3% sucrose, 0.8% agar, pH5.7, 1 mg/l BA (6-benzyladenine). The plates were layered with 1.5 ml ofmedia containing MS salts, B5 vitamins, 3% sucrose, pH 5.7, 4 mg/lp-chlorophenoxyacetic acid, 0.005 mg/l kinetin and covered with sterilefilter paper.

[0287] Following a 2.3 day co-culture, explants were transferred to deepdish petri plates (seven explants per plate) containing MS salts, B5vitamins, 3% sucrose, 0.8% agar, pH 5.7, 1 mg/l BA, 500 mg/lcarbenicillin, 50 mg/l cefotaxime, 200 mg/l kanamycin or 175 mg/lgentamicin for selection, and transferred after three weeks to freshmedia, five explants per plate. Explants were cultured in a growth roomat 25° C. with continuous light (Cool White). After an additional threeweeks, shoots were excised from the explants, and leaf recallusingassays were initiated to confirm modification of R₀ shoots. Three tinypieces of leaf tissue were placed on recallusing media containing MSsalts, B5 vitamins, 3% sucrose, 0.8% agar, pH 5.7, 5 mg/l BA, 0.5 mg/lnaphthalene acetic acid (NAA), 500 mg/l carbenicillin, 50 mg/lcefotaxime, 200 mg/l kanamycin or gentamicin or 0.5 mM glyphosate. Theleaf assays were incubated in a growth room under the same conditions asexplant culture. After an additional three weeks, the leaf recallusingassays were scored for herbicide tolerance (callus or green leaf tissue)or sensitivity (bleaching).

[0288] Each shoot stem was dipped in ROOTONE at the time of excision,placed in a two inch pot containing Metro-MIX 350, and maintained in aclosed humid environment in a growth chamber at 24° C., 16/8 hourphotoperiod, 400 uE per square meter per second (HID lamps) for ahardening-off period of approximately three weeks.

[0289] Plasmid pMON17138 is an Agrobacterium mediated single borderplant transformation vector maintained in the bacterium by selection onstreptomycin or spectinomycin. pMON17138 contains a single right Tiborder flanking the 3′ end of the genetic elements desired to betransferred into the plant genome. This vector contains two plantoperable expression cassettes. One cassette is comprised of acaulimovirus 35S promoter driving expression of a neomycinphosphotransferase gene (nptII), flanked downstream by a nopalinesynthase 3′ transcription termination and polyadenylation sequence (NOS3′). The other cassette is comprised of a figwort mosaic virus promoter(described in Rogers, U.S. Pat. No. 5,678,319) upstream of a pearibulose bisphosphate carboxylase small subunit transcriptiontermination and polyadenylation sequence. A chloroplast targetedglyphosate oxidoreductase (GOX) coding sequence is inserted between thepromoter and pea 3′ sequence.

[0290] Plasmid pMON17261 is an Agrobacterium mediated double borderplant transformation vector similar to pMON17138. A chloroplast targetedGOX encoding cassette identical to that in pMON17138 is presentdownstream from a Ti right border, and upstream of an additional plantoperable expression cassette comprised of a figwort mosaic viruspromoter (P-FMV) linked to a NOS 3′ sequence. A chloroplast targetedPhnO coding sequence is inserted between the second P-FMV and NOS3′sequences.

[0291] R₁ plants derived from transformation events using pMON17261 andpMON17138 were evaluated using a glyphosate spray test described inBarry et al. (U.S. Pat. No. 5,633,435).

[0292] Corn

[0293] An AMPA acyltransferase gene has also been introduced into BlackMexican Sweet corn cells with expression of the gene and glyphosateresistance detected in callus. Callus tissue was transformed accordingto the method described in Barry et al. (U.S. Pat. No. 5,463,175).Various plasmids were used to introduce glyphosate resistance genesencoding GOX and EPSPS in combination with an AMPA acyltransferase geneinto corn cells. These plasmids differed from each other with respect topromoters used, chloroplast or plastid targeting peptide sequences used,untranslated leader sequences used, presence or absence of an intron,and type of 3′ terminator used, however all plasmids contained asynthetically derived AMPA acyltransferase gene encoding PhnO containingthe P2A mutation. The synthetic gene was constructed from three smallerpolynucleotide sequences synthesized for Monsanto and characterized forthe presence of the desired DNA coding sequence and amino acid sequencetranslation by Stratagene, Inc., La Jolla, Calif. The non-naturallyoccurring gene was assembled from three smaller sequences comprised ofSEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, wherein the fullyassembled gene is represented by SEQ ID NO: 19, and is present in eachof the plasmids used for the corn callus transformation. Thenon-naturally occurring gene coding sequence was established based onthe method described in Fishhoff et al. in U.S. Pat. No. 5,500,365 inwhich monocot preferred codons were used in place of those preferred byE. coli. The fully assembled gene encodes a full length PhnO proteinidentical to the native protein sequence with the exception of the P2Amutation introduced by PCR using SEQ ID NO: 5 and SEQ ID NO: 6 toengineer appropriate restriction endonuclease recognition sites into theflanking ends of the coding sequence. Plasmids which were used ingenerating the corn callus data are shown in Table 15 along withdifferences with respect to genetic elements flanking the AMPAacyltransferase encoding sequence. TABLE 15 Corn Callus TransformationPlasmids and Relevant Genetic Elements Plasmid Relevant GeneticElements* pMON32926 [Pe35S/I-Zm.Hsp70/CTP/phnO/T-At.Nos] → GOX → EPSPSpMON32931 [Pe35S/I-Zm.Hsp70/   phnO/T-At.Nos] → GOX → EPSPS pMON32932[Pe35S/I-Zm.Hsp70/CTP/phnO/T-At.Nos] → GOX → EPSPS pMON32936[P-Os.Act1/I-Os.Act1/CTP/phnO/T-At.Nos] → GOX → EPSPS pMON32938[P-Os.Act1/I-Os.Act1/CTP/phnO/T-At.Nos] → GOX → EPSPS pMON32946[Pe35S/L-Ta.Cab/CTP/phnO/T-Ta.Hsp70]] → GOX → EPSPS pMON32947[Pe35S/L-Ta.Hsp70/CTP/phnO/T-Ta.Hsp70 ]] → GOX → EPSPS pMON32948 EPSPS →[Pe35S/I-Zm.Hsp70/CTP/phnO/T-At.Nos] → GOX pMON32950 EPSPS →[Pe35S/I-Zm.Hsp70/CTP/phnO/T-At.Nos] → GOX pMON32570 EPSPS →[Pe35S/L-Ta.Cab/I-Os.Act1/CTP/phnO/T-Ta.Hsp70] → GOX pMON32571 EPSPS →[Pe35S/L-Ta.Cab/I-Os.Act1/CTP/phnO/T-Ta.Hsp70] → GOX pMON32572 EPSPS →[Pe35S/L-Zm.Hsp70/I-Os.Act1/CTP/phnO/T-Ta.Hsp70] → GOX pMON32573 EPSPS →[Pe35S/L-Ta.Cab/I-Os.Act1/CTP/phnO/T-Ta.Hsp70] → GOX

[0294] Promoters which were used included the CaMV e35 S promoter andthe rice actin promoter (P-Os.Act1). Introns which were used includedthose obtained from plant genes such as corn Hsp70 (I-Zm.Hsp70) and riceactin (I-Os.Act1). Non-translated leader sequences which were usedincluded wheat chlorophyll a/b binding protein (L-Ta.Cab) and corn Hsp70(L-Zm.Hsp70). Termination and polyadenylation sequences which were usedincluded Agrobacterium tumefaciens NOS 3′ (T-At.Nos) and wheat Hsp70(T-Ta.Hsp70). The same chloroplast targeting sequence was used in allPhnO expression cassettes, represented by SEQ ID NO: 9.

[0295] A [¹⁴C]-glyphosate metabolism assay was used for determiningwhether transformed corn callus tissues contain functioning forms ofthese enzymes. The assay was developed to screen large numbers of corncallus samples. Callus was obtained from Monsanto Company and DekalbSeed Company corn transformation groups. The Monsanto callus samples,individually designated as callus lines “19nn-nn-nn” in Table 16, wereproduced from HI II×B73 corn embryos. Callus samples were bombarded withcomplete covalently closed circular recombinant plant transformationvector plasmid DNA or with linear DNA fragments isolated from suchplasmids 25-50 days after embryo isolation. Transformed lines wereidentified 8-14 weeks after bombardment. These lines were sub-culturedon fresh media every 2 weeks and were 5-7 months old when used in themetabolism assay. The Dekalb callus lines OO, OR, OW, OX, and OY wereobtained from HI II×AW embryos. All line designations correspond to therecombinant plasmid or linear fragment used for ballistic transformationof callus tissue as noted in the legend to Table 16.

[0296] 4.5 mCi of N-phosphono-[¹⁴C]-methylglycine ([¹⁴C]-glyphosate) wasobtained from the Monsanto Radiosynthesis group in a 1.5 mM aqueoussolution, having a specific radioactivity of 39.4 mCi/mM (5.2×10⁵dpm/microgram). The sample was identified with code number C-2182.2. Astock solution sterilized by filtration through a 0.2-micron Acrodisk(Gelman no. 4192) was prepared by combining 2.5 mL [¹⁴C]-glyphosate(3.3×10⁸ dpm) with 2.5 mL of corn callus growth medium (N6 medium) and5.0 mg of Mon 0818 surfactant. [¹⁴C]-glyphosate in the resulting dosesolution was 0.75 mM. The N6 medium was described by Chu et al. (1975)and was prepared using salts and vitamins obtained from Sigma ChemicalCompany, St. Louis, Mo. Mon 0818 surfactant is ethoxylated tallowamine,the surfactant used in Roundup herbicide. The dose solution wassubjected to HPLC analysis as described in Example 2. The results areshown in a chromatogram illustrated in FIG. 1. Three radioactive peakswere resolved, the largest of which corresponded to glyphosate (11.3min, 98.8%). Impurity peaks corresponding to [¹⁴C]-AMPA (5.8 min, 0.16%)and an unidentified material (10.2 min, 1.0%) were also present in thedose solution. No peaks corresponding to N-acetyl-[¹⁴C]-AMPA werepresent in the dose solution. Two additional dose solutions wereprepared using these reagents, each of which were scaled three fold to15 ml volumes based on the preparation method described above.

[0297] N-acetyl-[¹⁴C]-AMPA was synthesized for use as a retention timeHPLC standard. 1 mL of pyridine and 2 mL of acetic anhydride was addedto a 20-mL screw cap culture tube and chilled on ice. 0.1 mL of anaqueous solution of [¹⁴C]-AMPA (6.2×10⁶ dpm, code C-2127.2) was added tothe chilled solution. The tube was then removed from the ice bath andwarmed to about 50-60° C. A 10-μL sample was removed after about 30minutes and combined with 0.5 mL of water and analyzed according to theHPLC method set forth above. [¹⁴C]-AMPA was not detected, however twonew radioactive peaks were identified; one peak at 13.9 minutes (68%)and the other at 15.4 minutes (32%). A sample of the material eluting at13.9 minutes was isolated and analyzed by negative ion electrospray massspectrometry. The result showed strong ions at m/e 152 and 154, asexpected for this compound, which has a molecular weight of 153 Daltons;the m/z 154 ion was due to the isotopic [¹⁴C]atom. The radioactive peakeluting at 15.4 minutes was not isolated. However, in a separate HPLCexperiment, it was shown to co-elute with syntheticN-acetyl-N-methyl-AMPA. N-methyl-[¹⁴C]-AMPA has previously been shown tobe an impurity in the initial [¹⁴C]-AMPA material.

[0298] Under aseptic conditions, corn callus samples were transferred toindividual wells of sterile 48-well COSTAR™ cell culture clusters (cat.No. 3548). The individual callus samples were not weighed. However, inseveral cases the total weight of the callus samples in a 48-well platewas determined. Typically, the average weight of individual callussamples was approximately 200-250 mg. In each assay, a nontransformedcallus sample, HI II×B73, was included as a control. 50 μL of dosesolution containing 3.3×10⁶ dpm of [¹⁴C]-glyphosate was added to eachcallus sample. 48-well plates were sealed with parafilm and placed in aplastic bag containing a wet paper towel to provide a moist atmosphere.Bags were closed and placed in a dark drawer at 25° C. for 10 days. Eachcallus sample was subsequently transferred to a labeled microcentrifugetube (VWR, 1.7-mL, cat. No. 20170-620). 1.0 mL of de-ionized water wasadded to each tube, and the tubes were closed and placed in round20-tube floating microcentrifuge racks (Nalge cat. no. 5974-1015). Thesemicrofuge tubes were floated in boiling water for 30 minutes, shakenusing a vortex mixer, and centrifuged for 5 minutes using a Fisher brandmicrocentrifuge. 120-μL supernatant samples were removed for analysis byHPLC as described below. The samples were injected using a Waters WISPautoinjector. Chromatographic profiles were obtained for each sampleanalyzed, and quantitative information was obtained by extrapolating thearea under the radioactive elution peaks to total [¹⁴C] in each sample.FIG. 2 shows an HPLC profile of a mixture of standards of the observedradioactive metabolites [¹⁴C] AMPA, [¹⁴C] glyphosate, andN-acetyl-[¹⁴C]-AMPA and the impurity identified asN-acetyl-N-methyl-[¹⁴C]-AMPA.

[0299] HPLC analysis was typically completed using a SPHERISORB™ S5 SAX250 mm×10 mm column for most analyses. Some samples were analyzed on anALLTECH™ 5-micron, 250×10 mm SAX column, which provided similarperformance. Two solvents were prepared. Solvent A consisted of 0.005 MKH₂PO₄, adjusted to pH 2.0 with H₃PO₄ and contained 4% methanol. SolventB consisted of 0.10 M KH₂PO₄, adjusted to pH 2.0 with H₃PO₄ and alsocontained 4% methanol. The eluent flow rate was set at 3 mL/min, and thescintillation fluid flow rate was set at 9 mL/min using ATOMFLOW™scintillation fluid (No. NEN-995, from Packard Instruments). All columnsolvent steps were linear, with the injection and column solvent flowrates as indicated in example 2. The column is prepared for anadditional injection at 20 minutes.

[0300] Callus samples from 359 transformed corn lines were combined with50-μL aliquots of [¹⁴C]-glyphosate dose solution and incubated for 10days in the dark. Each post-incubation callus sample, together with itsclinging dose material, was transferred to a 1.7-mL microcentrifuge tubealong with 1 mL of water, and each tube was placed in boiling water.This step causes cell lysis, releasing soluble intracellular compoundsincluding any isotope labeled compounds such as glyphosate, AMPA, andN-acetyl-AMPA. It was determined during method development that if thepost-incubation calli were rinsed thoroughly with water, 85-95% of theradioactivity was rinsed off, and HPLC analysis showed that virtuallyall of the radioactivity in the rinses was due to [¹⁴C]-glyphosate andnone was attributable to [¹⁴C]-metabolites. In these experiments, therinsed calli gave extracts containing [¹⁴C]-metabolites in addition to[¹⁴C]-glyphosate. This indicated that the radioactivity in the rinseswas due mainly, if not exclusively, to unabsorbed surface[¹⁴C]-glyphosate. It is important to take this into account whenconsidering the rather low percentages of the dose converted tometabolites, because the percentage calculation includes large amountsof unabsorbed surface radioactivity. The method development work alsoshowed that simply boiling the incubated calli in water released as muchradioactivity as could be released by a conventional grinding/extractingprocedure. Experiments were conducted to show that oiling did not alterthe metabolite profiles. The streamlined procedures made it possible toanalyze large numbers of samples (e.g., 96) at one time. Table 16 showsrepresentative data of the callus samples producing the highest levelsof N-acetyl-[¹⁴C]-AMPA or [¹⁴C]-AMPA obtained after HPLC analysis. Arepresentative chromatogram of a GOX plus AMPA acyltransferasetransformed, glyphosate treated, callus extract sample is shown in FIG.3. TABLE 16 Transformed Corn Callus Lines Producing Amounts of AMPA orN-Acetyl-AMPA Callus Producing N-Acetyl-[¹⁴C]-AMPA Callus Producing[¹⁴C]-AMPA Transformed Percent ** Transformed Percent ** Callus * with .. . N-Acetyl-[¹⁴C]-AMPA Callus * with . . . [¹⁴C]-AMPA 1978-05-02pMON32570 0.27 1980-28-03 pMON32571 2.89 1978-08-01 pMON32570 0.94 OR523pMON32926 2.00 1978-20-02 pMON32570 0.57 OR534 pMON32926 5.00 1978-21-02pMON32570 0.23 OR537 pMON32926 2.00 1978-22-01 pMON32570 0.90 OR539pMON32926 5.08 1978-24-02 pMON32570 1.80 1971-08-01 pMON32932 2.641978-35-01 pMON32570 0.22 1971-27-03 pMON32932 3.63 1980-01-01 pMON325700.27 OO505 pMON32932 2.73 1980-03-01 pMON32571 0.22 OO509 pMON32932 2.861981-28-01 pMON32571 0.25 OO510 pMON32932 2.34 1981-02-01 pMON32572 0.65OO512 pMON32932 2.31 1981-03-01 pMON32572 0.74 OO514 pMON32932 1.981981-18-01 pMON32572 0.22 OO535 pMON32932 2.88 1981-23-01 pMON32572 0.48OO538 pMON32932 2.70 1981-24-02 pMON32572 0.29 OO539 pMON32932 1.971981-32-02 pMON32572 1.08 OO553 pMON32932 3.56 1977-05-03 pMON32573 0.39OO576 pMON32932 3.49 OR516 pMON32926 1.91 OO579 pMON32932 2.851972-14-01 pMON32931 0.40 1986-17-01 pMON32936 2.29 1972-32-01 pMON329310.75 1986-18-03 pMON32936 3.05 1972-33-01 pMON32931 0.55 1986-18-04pMON32936 2.15 OO544 pMON32932 0.28 1986-28-02 pMON32936 2.06 1986-06-01pMON32936 0.30 1983-12-02 pMON32938 2.41 1986-08-01 pMON32936 1.131983-31-01 pMON32938 2.90 1986-08-03 pMON32936 0.70 1985-03-02 pMON329462.51 1986-12-01 pMON32936 0.33 1985-38-01 pMON32947 1.99 1986-18-02pMON32936 0.40 OX512 pMON32948 2.43 1986-18-03 pMON32936 0.51 OX533pMON32948 3.91 1986-18-04 pMON32936 1.09 OX556 pMON32948 12.11 1986-22-04 pMON32936 0.64 OY504 pMON32950 2.25 1983-11-01 pMON32938 0.21OY511 pMON32950 2.53 OW534 pMON32946 0.77 OY528 pMON32950 2.58 OW542pMON32946 0.85 OY532 pMON32950 2.24 1985-26-01 pMON32947 0.60 OY534pMON32950 4.02 1985-26-03 pMON32947 0.71 OY535 pMON32950 2.34 1985-11-04pMON32952 0.37 OY540 pMON32950 5.57

[0301] 19 of the 359 callus samples tested produced extracts containingN-acetyl-[¹⁴C]-AMPA at a level distinctly higher than the other callussamples. Callus OR516 was the strongest in this respect and was analyzedfive times during a period of two months, providing values ranging from0.50-4.54% (average 1.91%). The basis for the relatively large spread inthe percentage of N-acetyl-[¹⁴C]-AMPA formed at various times isunknown. In four of the five analyses of OR516, the percentage ofN-acetyl-[¹⁴C]-AMPA present was higher than that of [¹⁴C]-AMPA,indicating an efficient conversion of [¹⁴C]-AMPA to N-acetyl-[¹⁴C]-AMPA.The callus next most efficient in producing N-acetyl-[¹⁴C]-AMPA was1978-24-02, which was the only other callus besides OR516 that containedmore N-acetyl-[¹⁴C]-AMPA than [¹⁴C]-AMPA in its extract. One hundred ofthe 359 callus samples tested produced extracts containing [¹⁴C]-AMPA ata level distinctly higher than other callus samples. OX556 was asuperlative producer of [¹⁴C]-AMPA, yielding more than twice as much ofthe metabolite as any other callus in the study. The control callus, HIII×B73, which contained no inserted genes, produced no detectable levelsof N-acetyl-[¹⁴C]-AMPA and only background levels of [¹⁴C]-AMPA. Thisresult indicates that expression of an AMPA acyltransferase in corn iseffective in conversion of AMPA produced as a result of GOX mediatedglyphosate degradation to N-acetyl-AMPA.

[0302] Wheat

[0303] GOX mediated glyphosate degradation has been shown to produceAMPA, and AMPA has previously been shown to be the source of phytotoxiceffects. Therefor, effects of wheat plant exposure to the compounds AMPAor N-acetyl-AMPA was determined as in example 2 in order to observe anywheat sensitivity or insensitivity to either of these compounds. Theobservation of any phytotoxic effects would indicate that GOX mediatedglyphosate metabolism would be detrimental to Triticum species.

[0304] Wheat immature embryos were exposed to different concentrationsof AMPA and N-acetyl-AMPA in a wheat embryo germination assay. MMSO basemedia was prepared containing 40 grams per liter maltose, 2 grams perliter GELRITE™, MS salts, and vitamins. Salts, vitamins, and maltosewere dissolved in 3500 ml water and the pH was adjusted to 5.8. 500 mlwas dispensed into a separate bottle along with 1 gram of GELRITE™ andautoclaved for 17 minutes. After the medium had cooled to about 45° C.,AMPA or N-acetyl-AMPA was added to a defined concentration. The mixturewas poured into six square Sundae cups under sterile conditions andallowed to solidify.

[0305] Immature wheat embryos were isolated from twenty day oldseedlings (after anther formation) and inoculated into each MMSO media.Each Sundae cup contained nine immature embryo's. Three separate plateswere used for each concentration of AMPA (0, 0.1, 0.15, 0.2, 0.25, 0.3,and 1.0 mM) or N-acetyl-AMPA (0, 0.1, 0.3, 1.0, and 3.0 mM). Sundae cupswere incubated for ten days and the length of roots and shoots weredetermined and compared. The results are shown in Table 17. TABLE 17Comparison of AMPA and N-acetyl AMPA on Germinating Shoot and RootLength Phosphonate Compound Shoot (cm) Root (cm) AMPA (mM) 0.00 12.6 ±2.6 7.0 ± 1.9 0.10 11.7 ± 2.5 8.0 ± 2.0 0.15 11.3 ± 2.1 6.3 ± 1.7 0.20 9.2 ± 1.8 4.6 ± 2.1 0.25  8.5 ± 1.8 3.1 ± 1.6 0.30  6.6 ± 1.8 2.6 ± 1.61.00  0.9 ± 0.1 0.4 ± 0.1 N-Acetyl-AMPA 0.00 12.6 ± 2.6 7.0 ± 1.9 0.1012.0 ± 2.4 5.9 ± 1.4 0.30 11.7 ± 3.5 5.2 ± 1.2 1.00 12.2 ± 3.2 5.4 ± 1.53.00 11.2 ± 2.6 5.9 ± 1.6

[0306] AMPA was not substantially inhibitory to growth and elongation ofimmature embryo's at concentrations under 0.2 mM. However,concentrations above 0.2 mM were severely inhibitory to both shoot androot elongation, indicating that AMPA may also be phytotoxic to wheatand, considering the nature of the monocot crop species as a whole,phytotoxic to other monocotyledonous crops as well as turf grasses.Germination of immature embryo's was significantly affected when theAMPA level was higher than 0.20 mM. 1.00 mM AMPA eliminated thegermination of immature embryo's in wheat. In contrast, N-acetyl-AMPAwas not inhibitory to shoots and only mildly inhibitory to rootelongation at any concentrations tested in this experiment. The highestN-acetyl-AMPA concentration tested was greater than ten times theminimal non-inhibitory concentration determined for AMPA. There are nosignificant effects to immature embryo germination when theN-acetyl-AMPA concentration is less than 3.0 mM. This result indicatesthat N-acetylation of AMPA in wheat would prevent AMPA phytotoxicityarising as a result of GOX mediated glyphosate herbicide metabolism.

[0307] Recombinant glyphosate tolerant wheat plants were generatedaccording to the method of Zhou et al. (Plant Cell Reports 15:159-163,1995). Briefly, spring wheat, Triticum aestivum cv Bobwhite, was used asthe target transformation line. Stock plants were grown in anenvironmentally controlled growth chamber with a 16 hour photoperiod at800 microJoule per square meter per second provided by high-intensitydischarge lights (Sylvania, GTE Products Corp., Manchester, N.H.). Theday/night temperatures were 18/16° C. Immature caryopses were collectedfrom the plants 14 days after anthesis. Immature embryos were dissectedaseptically and cultured on MMS2 medium, a Murashige and Skoog (Physiol.Plant 15:473-497, 1962) basal medium supplemented with 40 grams perliter maltose and 2 milligrams per liter 2,4-D. In some experiments, CM4medium was used. CM4 medium contains is MMS2 medium, but contains only0.5 milligrams per liter 2,4-D and includes 2.2 milligrams per literpicloram. The immature embryos were cultured at 26° C. in the dark.

[0308] Immature embryos were transferred five days after cultureinitiation to an osmotic treatment CM4 medium containing 0.35 M mannitolfour hours prior to bombardment according to the method of Russell etal. (In Vitro Cell Devel. Biol., 28P:97-105, 1992). Thirty to fortyembryos were placed in the center of each plate and bombarded in aDuPont PDS1000 apparatus. Plasmid DNA was adsorbed onto 1 μm tungstenparticles according to the method of Sanford et al. (Particle Sci.Technol., 5:27-37, 1987). Embryos were bombarded twice at a distance of13 mm from the stopping plate. A 100 μm stainless steel screen wasplaced immediately below the stopping plate.

[0309] After a 16 hour post bombardment treatment on the osmotic medium,the embryos were transferred to MMS2 or CM4 medium. Following a one weekdelay, the embryos were transferred to the MMS2 or CM4 medium containing2 mM glyphosate. After 9-12 weeks of callus proliferation on theselection medium, calli were transferred to a MMS.02 regeneration mediumcontaining 0.2 mg/l 2,4-D and 0.1 mM glyphosate. Shoots obtained fromthe regeneration medium were transferred to MMSO without 2,4-D butcontaining 0.02 mM glyphosate.

[0310] Glyphosate tolerant R₀ plants as well as R₁ progeny weretransferred to 15 centimeter diameter pots and grown in anenvironmentally controlled chamber as described above. Two weeks later,the plants were sprayed with 3 ml/liter ROUNDUP (41% active ingredient,Monsanto Company) in a spray chamber, which was designed to mimic afield dose application of 0.6 kilograms glyphosate per hectare. Damagesymptoms were observed and recorded at different stages following thespraying.

[0311] Genomic DNA was isolated from leaf tissue of R₀ and R₁ progenyfollowing the method of Shure et al. (Cell 35:225-233, 1983). Fifteenmicrograms of genomic DNA was digested with BglII restrictionendonuclease and fractionated on a 0.8% agarose gel. The DNA wastransferred to Hybond N membranes (Amersham) according to the standardprocedure described in Sambrook et al. (Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press, 1989). The membranes wereprobed independently for the presence of genes encoding EPSPS and GOX. A3.4 kb DNA fragment containing the EPSPS gene and a 4.8 kb DNA fragmentcontaining the GOX gene were released from pMON19574 by BglIIrestriction endonuclease digestion, isolated by 0.7% agarose gelelectrophoresis, and labeled with [³²p] dCTP using a Stratagene PRIME-ITII random primer labeling kit. Probes were labeled to a specificactivity of 3×10⁹ counts per minute per microgram and 1.3×10⁹ counts perminute per microgram, respectively. Membranes were hybridized for 14hours at 42° C. in a solution containing 50% formamide, 5×SSC,5×Denhardt's, 0.5% SDS, and 100 microgram per milliliter tRNA. Thecondition of the final wash was 0.1% SSC and 0.1% SDS at 60° C. forfifteen minutes.

[0312] EPSPS and GOX protein assays were conducted using crude proteinextracts from leaf tissue of R₀ plants and total proteins werequantified following the method of Bradford (Anal. Biochem. 72:248-256,1976). The percentage of EPSPS and GOX protein represented in theextracts was quantified using an ELISA method and calculated as percenttotal extractable protein.

[0313] Immature embryos from the R₀ transgenic and Bobwhite controlplants were isolated twenty days after anthesis and cultured on the MMS0medium with 0.02 mM glyphosate for a germination test. Germinated andnon-germinated embryos were recorded ten days later and the data wasanalyzed by χ² test for 3:1 segregation. Tolerant plants from thegermination test were transferred to soil and sprayed with threemilliliters per liter of ROUNDUP as described above.

[0314] Five plasmids harboring glyphosate resistance genes were used totransform immature wheat embryos as described above. pMON19338 containsa nucleotide cassette encoding a petunia EPSPS chloroplast transitpeptide in frame with an Agrobacterium strain CP4 EPSPS enzyme sequence.The nucleotide cassette is inserted downstream of a cauliflower mosaicvirus enhanced 35S promoter linked 3′ to a maize HPS70 intron sequenceand upstream of a nopaline synthase 3′ transcription termination andpolyadenylation sequence. Convenient restriction sites are positionedbetween the intron sequence and the 3′ termination sequence forinsertion of genetic elements. pMON19643 is identical to pMON19338except that a GOX enzyme encoding sequence is used in place of theAgrobacterium EPSPS enzyme encoding sequence. pMON19574 is identical topMON19338 but additionally contains a chloroplast targetedglyphosate-oxidoreductase expression cassette identical to that inpMON19643 downstream of and immediately adjacent to the EPSPS expressioncassette. pMON32570 is similar to pMON19574 in that expression cassettesencoding a chloroplast targeted EPSPS and chloroplast targeted GOX arepresent, however, an expression cassette encoding a chloroplast targetedAMPA acyltransferase enzyme is also present between the EPSPS and GOXexpression cassettes. Other elements which are present in pMON19574 andnot in the other plasmids are also worthy of mention. For example, awheat major chlorophyll a/b binding protein gene 5′ untranslated leaderis present between the enhanced 35S promoter and intron in both theEPSPS and AMPA acyltransferase expression cassettes (McElroy et al.,Plant Cell 2:163-171, 1990). Also, a wheat hsp17 gene 3′ transcriptiontermination and polyadenylation sequence is present in place of thenopaline synthase 3′ sequence for both EPSPS and AMPA acyltransferaseexpression cassettes. All plasmids produced recombinant glyphosatetolerant wheat plants using the ballistic transformation methoddescribed above. However, plasmids which were capable of expressing GOXonly or GOX along with an AMPA acyltransferase either did not producerecombinant glyphosate tolerant wheat plants or produced plants whichexperienced problems with stunted growth, aberrant segregation ofphenotypes, and infertility and were not analyzed further. The dataobtained after biolistic transformation using the described plasmids isshown in Table 18. TABLE 18 Wheat Biolistic Transformation DataGlyphosphate Tolerance # Transgenic Transformation Gene(s) # ExplantsEvents Efficiency¹ GOX 120 0 0 GOX + PhnO 434 6 1.4 EPSPS 120 6 5.0EPSPS + GOX 120 1 0.8 EPSPS + PhnO + GOX 10,068 314 3.1

[0315] Transformed glyphosate tolerant plants arising out of thesetransformations were self crossed and allowed to produce R1 seed, whichwere used to generate R1 plants. Glyphosate tolerance generallysegregated in the expected ratio of 3:1 in R1 plants as judged by R1plant sensitivities after spraying with glyphosate at the three leafstage. Glyphosate tolerant R1 plants were self crossed and allowed toproduce R2 seed. R2 seed was germinated from a number of differentglyphosate tolerant lines to produce R2 glyphosate tolerant plants towhich [¹⁴C]-glyphosate was applied as described above. Plant leaf andstem tissues were harvested at 48 hours after glyphosate application,and water soluble compounds were extracted as described above andanalyzed by HPLC as in example 2 for the presence of [¹⁴C]-glyphosatemetabolites. The total area under the [¹⁴C] isotope labeled peakseluting from the column was summed to provide a baseline of 100%[¹⁴C]-compound identification for each sample analyzed. The results areshown in Table 19. TABLE 19 Glyphosate Metabolism In Wheat PlantExtracts¹ Sample & Glyphosate Plant [¹⁴C]- [¹⁴C]- Acetyl- ToleranceGene(s) Line No. Glyphosate AMPA [¹⁴C]-AMPA [¹⁴C]-Other⁴ Standard² na 3026 31 13 na 29 24 29 18 na 35 29 36 0 Growth Medium³ na 60 32 0.2 8 na48 25 2 25 na 87 7 0 6 EPSPS 24756 43 25 1 31 24756 53 46 0 1 25397 6138 0 1 25397 37 19 1.2 43 25397 64 20 0 16 EPSPS + PhnO + GOX 27249 6 785 2 27249 14 12 61 13 27249 5 24 33 38 25462 48 21 0 31 25462 44 5 0 5125462 54 35 0 11 26281 48 14 17 21 26281 64 11 13 12 26281 38 7 7 4828598 20 7 5 68 28598 25 7 5 63 Bobwhite na 74 26 0 0 na 17 15 0 32 na34 24 0 42

[0316] The standard solution contains approximately equal molar ratiosof each of the compounds glyphosate, AMPA and N-acetyl-AMPA, as well asa number of impurities which are present as a result of the chemicalsynthesis of these isotope labeled compounds. Growth medium to which[¹⁴C]-glyphosate was added was treated to the same conditions as wheatplants, ie, the medium was exposed to incident light intensities whichplants received. As expected, photodegradation of glyphosate to AMPA wasobserved, and a small percentage of AMPA appeared to be converted toacetyl-AMPA, probably as a result of exposure in the growth medium toother acylated compounds. Photodegradation of glyphosate by visiblelight exposure to AMPA as the major degradation product has beenobserved previously (Lund-Hoeie et al., Photodegradation of theherbicide glyphosate in water. Bull. Environ. Contam. Toxicol.36:723-729, 1986). Recombinant wheat plants transformed with anEPSPS-only plasmid did not produce [¹⁴C]-AMPA or acetyl-[¹⁴C]-AMPA from[¹⁴C]-glyphosate. [¹⁴C]-AMPA and trace amounts of acetyl-[¹⁴C]-AMPAwhich were observed were within the limits observed as a result ofphotodegradation in the growth medium control. Non-recombinant Bobwhitecontrol plants treated with [¹⁴C]-glyphosate also did not produce AMPAor acetyl-AMPA. Plants transformed with the triple gene constructplasmid containing genes capable of expressing EPSPS, PhnO and GOXproduced variable results. About one third of these plants appeared toefficiently convert glyphosate to acetyl-AMPA, indicating that the GOXand PhnO enzymes were present and functional. Southern blot analysesdemonstrated that the transgenes were integrated into the wheat genomesand transmitted to the following generations. Western blot analysisusing anti-EPSPS, anti-GOX, or anti-PhnO antiserum to detect theseproteins in the triple gene transformed plant extracts provided furtherinsight into the basis for the variable [¹⁴C]-glyphosate metabolismobservation. Western blot analysis indicated that all of the lines wereproducing EPSPS, however only line 27249 was producing GOX and PhnOprotein. This result is consistent with the data in Table 19, whichshows that line 27249 efficiently metabolizes [¹⁴C]-glyphosate toacetyl-[¹⁴C]-AMPA. This plant line also did not demonstrate stunting,partial fertility, or altered segregation phenotypes associated withother lines. These results indicate that co-expression of GOX and AMPAacyltransferase in wheat plants expressing recombinant EPSPS providesimproved herbicide tolerance.

Example 9

[0317] This example illustrates the transformation of tobaccochloroplasts with a phnO gene.

[0318] Recombinant plants can be produced in which only themitochondrial or chloroplast DNA has been altered to incorporate themolecules envisioned in this application. Promoters which function inchloroplasts have been known in the art (Hanley-Bowden et al., Trends inBiochemical Sciences 12:67-70, 1987). Methods and compositions forobtaining cells containing chloroplasts into which heterologous DNA hasbeen inserted have been described, for example by Daniell et al. (U.S.Pat. No. 5,693,507; 1997) and Maliga et al. (U.S. Pat. No. 5,451,513;1995). A vector can be constructed which contains an expression cassettefrom which an acyltransferase protein could be produced. A cassettecould contain a chloroplast operable promoter sequence drivingexpression of, for example, a phnO gene, constructed in much the samemanner as other polynucleotides herein, using PCR methodologies,restriction endonuclease digestion, and ligation etc. A chloroplastexpressible gene would provide a promoter and a 5′ untranslated regionfrom a heterologous gene or chloroplast gene such as psbA, which wouldprovide for transcription and translation of a DNA sequence encoding anacyltransferase protein in the chloroplast; a DNA sequence encoding anacyltransferase protein; and a transcriptional and translationaltermination region such as a 3′ inverted repeat region of a chloroplastgene that could stabilize an expressed mRNA coding for anacyltransferase protein. Expression from within the chloroplast wouldenhance gene product accumulation. A host cell containing chloroplastsor plastids can be transformed with the expression cassette and then theresulting cell containing the transformed chloroplasts can be grown toexpress the acyltransferase protein. A cassette may also include anantibiotic, herbicide tolerance, or other selectable marker gene inaddition to the acyltransferase gene. The expression cassette may beflanked by DNA sequences obtained from a chloroplast DNA which wouldfacilitate stable integration of the expression cassette into thechloroplast genome, particularly by homologous recombination.Alternatively, the expression cassette may not integrate, but byincluding an origin of replication obtained from a chloroplast DNA,would be capable of providing for replication of, for example, aheterologous phnO or other acyltransferase gene within the chloroplast.

[0319] Plants can be generated from cells containing transformedchloroplasts and can then be grown to produce seeds, from whichadditional plants can be generated. Such transformation methods areadvantageous over nuclear genome transformation, in particular wherechloroplast transformation is effected by integration into thechloroplast genome, because chloroplast genes in general are maternallyinherited. This provides environmentally “safer” transgenic plants,virtually eliminating the possibility of escapes into the environment.Furthermore, chloroplasts can be transformed multiple times to producefunctional chloroplast genomes which express multiple desiredrecombinant proteins, whereas nuclear genomic transformation has beenshown to be rather limited when multiple genes are desired.Segregational events are thus avoided using chloroplast or plastidtransformation. Unlike plant nuclear genome expression, expression inchloroplasts or plastids can be initiated from only one promoter andcontinue through a polycistronic region to produce multiple peptidesfrom a single mRNA.

[0320] The expression cassette would be produced in much the same waythat other plant transformation vectors are constructed. Plantchloroplast operable DNA sequences can be inserted into a bacterialplasmid and linked to DNA sequences expressing desired gene products,such as PhnO proteins or other similar acyltransferases, so that theacyltransferase protein is produced within the chloroplast, obviatingthe requirement for nuclear gene regulation, capping, splicing, orpolyadenylation of nuclear regulated genes, or chloroplast or plastidtargeting sequences. An expression cassette comprising a phnO or similaracyltransferase gene, which is either synthetically constructed or anative gene derived directly from an E. coli genome, would be insertedinto a restriction site in a vector constructed for the purpose ofchloroplast or plastid transformation. The cassette would be flankedupstreamn by a chloroplast or plastid functional promoter and downstreamby a chloroplast or plastid functional transcription and translationtermination sequence. The resulting cassette could be incorporated intothe chloroplast or plastid genome using well known homologousrecombination methods. Alternatively, chloroplast or plastidtransformation could be obtained by using an autonomously replicatingplasmid or other vector capable of propagation within the chloroplast orplastid. One means of effectuating this method would be to utilize aportion of the chloroplast or plastid genome required for chloroplast orplastid replication initiation as a means for maintaining the plasmid orvector in the transformed chloroplast or plastid. A sequence enablingstable replication of a chloroplast or plastid epigenetic element couldeasily be identified from random cloning of a chloroplast or plastidgenome into a standard bacterial vector which also contains achloroplast or plastid selectable marker gene, followed bytransformation of chloroplasts or plastids and selection for transformedcells on an appropriate selection medium. Introduction of an expressioncassette as described herein into a chloroplast or plastid replicableepigenetic element would provide an effective means for localizing anacyltransferase gene and protein to the chloroplast or plastid.

[0321] In view of the above, it will be seen that the several advantagesof the invention are achieved and other advantageous results attained.As various changes could be made in the above methods and compositionswithout departing from the spirit and scope of the invention, it isintended that all matter contained in the above description, and shownin the accompanying drawings and sequences, shall be interpreted asillustrative and not in a limiting sense.

[0322] Referenced Literature:

[0323] Avila et al., J. Am. Chem. Soc. 109:6758-6764, 1987.

[0324] Berlyn, Microbiol. Molec. Biol. Rev. 62:814-984, 1998.

[0325] Chen et al., J. Biol. Chem. 265:4461-4471, 1990.

[0326] Dumora et al., Biochim. Biophys. Acta 997:193-198, 1989.

[0327] Franz, Discovery, development and chemistry of glyphosate, in TheHerbicide Glyphosate. Eds. E. Grossbard and D. Atkinson. Butterworths.pp. 3-17, 1985.

[0328] Hanley-Bowden et al., Trends in Biochemical Sciences 12:67-70,1987.

[0329] Hilderbrand et al., The role of phosphonates in living systems;Hilderbrand, R. L., Ed, pp. 5-29, CRC Press, Inc., Boca Raton, Fla.,1983.

[0330] Jacob et al., Appl. Environ. Microbiol. 54:2953-2958, 1988

[0331] Jiang et al., J. Bacteriol. 177:6411-6421, 1995.

[0332] Kishore et al., J. Biol. Chem. 262:12,164-12,168, 1987.

[0333] Lacoste et al., J. Gen. Microbiol. 138:1283-1287, 1992.

[0334] Lee et al., J. Bacteriol. 174:2501-2510, 1992.

[0335] Maier, Phosphorous Sulfur 14:295, 1983.

[0336] Makino et al., J. Bacteriol. 173:2665-2672, 1991.

[0337] McGrath et al., Eur. J. Biochem. 234:225-230, 1995.

[0338] Metcalf et al., J. Bacteriol. 173:587-600, 1991.

[0339] Metcalf et al., Gene 129:27-32, 1993.

[0340] Ohtaki et al., Actinomyceteol. 8:66-68, 1994.

[0341] Pipke et al., Appl. Environ. Microbiol. 54:1293-1296,1987.

[0342] Shinabarger et al., J. Bacteriol. 168:702-707, 1986.

[0343] Tanaka et al., J. Fac. Agr. Kyushu Univ. 30:209-223, 1986.

[0344] Wackett et al., J. Bacteriol. 169:710-717, 1987a

[0345] Wackett et al., J. Bacteriol. 169:1753-1756, 1987b.

[0346] Wanner et al., FEMS Microbiol. Lett. 100: 133-140, 1992.

[0347] Wanner, Biodegradation 5:175-184, 1994.

[0348] Wohlleben et al., Mol. Gen. Genet. 217:202-208, 1989.

[0349] Referenced Patent Documents:

[0350] Barry et al., U.S. Pat. No. 5,463,175, 1995.

[0351] Barry et al., U.S. Pat. No. 5,633,435, 1997

[0352] Comai, U.S. Pat. No. 4,535,060, 1985.

[0353] Daniell et al., U.S. Pat. No. 5,693,507; 1997.

[0354] Maliga et al., U.S. Pat. No. 5,451,513; 1995.

[0355] McBride et al., WO 95/24492.

1 32 1 15611 DNA Escherichia coli 1 ggatccagca tcgacgccag tttttccaccattgtcagtc gcaggctaag cggcgcattt 60 aacatgccgc cgttcgtcca tgtctgaagctgcacacgcg aaagaagttc ctgcatcagt 120 cgttcacgaa actgctgctg atgggcttgtggaaggcggg catcatcgcc ctgcgccaga 180 tccactaaaa agcggggata aaccgactccagcacgcgac cggggccgtc cagtaacgtc 240 ttggtcaata tcgttctgcc gtgaaaagtgtttgaatatc atcgcgtaac agctgggcgt 300 cggtgtaaat ccagccgtga gtcatcacagtctgctgcaa ttgctgctgc atcagcctga 360 ccaccgattc attttgttga cgcagagccaggctttcgcg taaacgcgtc tgtaattccg 420 tcaaacatga agcgaactca gcgaaaaaagtattcatgcc tgccgtaaca gattcatcga 480 cctgctctgc cagaacttta gccatttgttggcaataaag atcgacttct gcgcttaatg 540 ctcgttgcaa cacactgtaa tcaaccgtttctgtcgggga tttctcattt ccccgtcccc 600 agtcgggctg attcaaccag cgcgaaaaagtctcacgcac aacgcctaaa cgcgtgctct 660 gctcgtccgt tgcatcctgg cgcgaaatgactgcactgaa cagctggcga gtgttgaagt 720 ggggaactac gccgtgaaaa acaggaaaatgaaacccagg acgaaaccct gactcgctca 780 attccatttt gacttgttgc tcaatggggcgaataacatc ggttaacact cggcaaaggg 840 tggattccag ctcggcaaaa cgcagcgtaaagtcgcgact gatggtgttc tgcgccgtct 900 gtaacagtgt ctcacagcgg gtacgcatctcgcttaacgg ctccgaatca tcctgaaaca 960 aggcggctaa ctgcgcattc agcgcatcttgttgttgacg cagaaagtgg ttggcggagg 1020 tcagggccag ctcgatttca tgtttaatctcgccgctcac ctgcgcctga ttgagttgca 1080 atagctgcaa actttcttcg acctgatggatattttgccg caattgttca caagcgacgt 1140 ttaacccgtg cgcacgaaaa tccaggtattcccgcgcctg ctgcgcgtaa ttcaacagtt 1200 tatgcgcagc agatcgcaaa gcatacaacgaggcgttagc gtaagcggca tgaagcaacg 1260 cctgaattgg ctgggcgaac agcgaatcttcccacaactg atcggcagca tgacgaatat 1320 gttcgaggtc cgccagatcg gcatgacgccagcgcctgcc gagcgcggca tgggcaaaat 1380 cttccaccca gcgttgttgc tctggcgctggtaacttacc gttgttggct aactcatggc 1440 gcgcccgatt cgccaggtag ccccacatcgacgacaccgg aaatatctgc tgtggcgtaa 1500 tacagccttt catcagcgtc ccggaaatcagtgcccgcac ctggtcggcg tcgtcactgt 1560 tacgatcctg ttgatcgaac ttattgaccagcacatacag cggcaccgat tgccccaccg 1620 ccaaaatcgc ctcacggacc tcttcatcggagatcgattt cagttgcgta taatccagca 1680 ccgccagtac cgccgaggcg cgtgccagctgctggttaag cattttttgc agatgcggtt 1740 gcccggcttc atttggcccg ggggtatccagtaacgtcaa ctgaccggga taactctcca 1800 gccccgccag atggacaaac tccacttcaatcacgggaat atgctcaatg gcggcgtaag 1860 cagaaaaagg aaaatcgacg tccagcgccttcgccagtcg cactaaatca ttcaaacttt 1920 tcagacaatg aaaaataggc tgggcacccagataatattt ttcgaaagcg acgccatttt 1980 cgatccgctg cataagcgca cgcatatctttatctatttc cagcacatcg gtcagatgct 2040 taatatcgca atcacgcagg cgctgttgtaattgttgaat taaacaatcg attggcgcga 2100 catgtgaaaa atgcagtacc ggttccttttgcccgggcgt atggcgaata agcgtcggca 2160 gcgcagtcat tgggcgatta cgattaggcagaacctccgt accaacaatg gcattaatgg 2220 tggttgattt ccctgctttc atggtaccgacaattgcaag caccatttcc agtcgggaaa 2280 ttttacgcaa ctcattattc agcatcgcgtgacgttcggc gatattaggc tgactccagg 2340 gtaaagccag ttgtggcgcg tcgtctccgggtacagagag aggcattttt tccagtaact 2400 gcaactgttg gcgagaaagc tgtaacaggcgttcagcctc ctgacttaac tcatacaggg 2460 tctgtgtgta catagaaaat tcttccttaaagcaaatttt gttattttat ttagccagat 2520 tgtttttgag ttctgttttc ggcttttataattactgcaa gaaataattt tatatttagt 2580 gtgttgtttt ttatcagaat aaataacgtcttctgatacg tttaaaacgt cagaaagata 2640 aaaatatcat gtgaattaaa aaaagaacaagtagagcatt aacattatct taaataataa 2700 atagaggcaa aaagattatt ttctttttgcgtttcctttc aaatgaaaac gatcgtcgtc 2760 taaaatcagc agtacccccg acaaactcagggattttgtg tataattgcg gcctttttcg 2820 gcaatctgcc gttttttggc gcttttgccctgctgacttt tgaggaaatc cacatgtcat 2880 taccacactg cccaaaatgc aactccgaatacacttacga agataacggc atgtacatct 2940 gcccggaatg tgcctacgaa tggaacgacgcagaacctgc acaggaaagc gacgagctga 3000 tcgttaaaga tgctaacggc aatctgctggctgacggcga cagcgttacc atcattaaag 3060 atctgaaggt gaaaggtagc tcttcgatgctgaaaattgg caccaaagtg aaaaacatcc 3120 gcctggttga aggcgaccat aacatcgattgcaaaatcga cggttttggt ccgatgaaac 3180 tgaaatctga gtttgtgaaa aagaactgattgtattgtga tcggtaagcc ggataaggcg 3240 ctcgcgccgc atccggcaac ggtgccagatgcctgatgcg acgcttgcgc gtcttatcag 3300 gcctacaaat tcccgcaccc tccgtaggccggataaggcg tttacgccgc atccggcaac 3360 ggtgccgact gcctgatgcg acgcttgcgcgtcttatcag gcctacaaat tcccgcaccc 3420 tccgtaggcc ggataaggcg tttacgccgcatccggcaac agtgccaact gcctgatgcg 3480 acgcttgcgc gtcttatcag gcctacaaattcccgcaccc tccgtaggcc ggataaggcg 3540 tttacgccgc atccggcaat ggtgccgactgcctgatgcg acgcttgcgc gtcttatcag 3600 gcctacaaat tcccgcaccc tccgtaggccggataaggcg tttacgccgc atccggcaac 3660 agtgccgact gcctgatgcg acgctcgcgcgtcttatcag gccgcctctc atctgtataa 3720 atttcgaact acacttaact ggcttctcttaactgaggtc accatcatgc cgttaagtcc 3780 ctacctctct tttgccggta actgttccgacgcgattgcc tattatcaac gtacgttggg 3840 cgcggaactg ctctataaaa tcagcttcggcgaaatgcca aaatcagcgc aggacagcgc 3900 cgagaactgc ccttccggaa tgcaatttcccgataccgcc atcgctcatg ccaacgtgcg 3960 cattgccgga agcgacatca tgatgagcgatgccatgccg tcaggaaaag ccagctactc 4020 cggctttacg ctggtgctcg attcgcaacaggtcgaagaa ggaaaacgct ggtttgacaa 4080 tcttgccgct aacggaaaaa tcgaaatggcctggcaggaa actttctggg cgcatggctt 4140 tggcaaagtc accgataaat ttggcgtaccgtggatgatt aatgtcgtca aacaacaacc 4200 aacgcaataa cccgccggga ggcccgccctcccgcactgt catcgaattc ccgttaactc 4260 ttcatctgtt agtcactttt aattaaccaaatcgtcacaa taatccgcca cgatggagcc 4320 acttttttag ggaggctgca tcatgcaaacgattatccgt gtcgagaagc tcgccaaaac 4380 cttcaatcag catcaggcgc tgcatgcggttgatctgaac attcatcacg gtgaaatggt 4440 ggctctgctt gggccgtcgg gttccggcaaatccaccctt ttacgtcact taagcggttt 4500 gattaccggc gataaatccg ccggcagccatatcgagctg ctgggccgca cagtccagcg 4560 cgaaggccgt ctggcgcgcg atatccgcaaaagccgcgcc aacaccggct acatcttcca 4620 acaattcaac ctggtgaacc gcctgagcgtactggagaac gtgctgattg gcgcgctcgg 4680 cagcacgccg ttctggcgca cctgttttagctggtttacc cgcgagcaga aacaacgcgc 4740 gttacaggcg ctgacccgcg ttggcatggtgcattttgcc catcaacgcg tttccaccct 4800 ctccggcgga cagcagcagc gtgtggcgattgcccgcgcg ctgatgcagc aggcgaaggt 4860 gattctggcc gatgaaccca tcgcctcgctggacccggaa tccgcccgca tcgtgatgga 4920 caccctgcgc gacatcaatc agaacgacggcatcaccgtg gtcgtcacgc tgcatcaggt 4980 ggattacgcc ctgcgctact gcgaacgcatcgtcgccctg cgccaggggc acgttttcta 5040 cgacggcagc agccaacagt ttgataacgaacgttttgac catctctacc gcagcattaa 5100 tcgcatcgaa gagaacgcga aagctgcctgacatccccat cattgaggaa aacgaatgaa 5160 cgctaagata attgcctcgc tggccttcaccagcatgttc agcctcagca ccctgttaag 5220 cccggcacac gccgaagagc aggaaaaggcgctgaatttc ggcattattt caacggaatc 5280 acagcaaaac ctgaaaccgc aatggacgccattcttacag gatatggaga agaagctggg 5340 cgtgaaggtg aacgccttct ttgccccagactacgcaggc attatccagg gaatgcgctt 5400 caataaagtg gatatcgcct ggtacggcaacctgtcggca atggaagcgg tggatcgcgc 5460 caacggccag gtcttcgccc agacggtcgcggcggatgga tcgccaggtt actggagcgt 5520 gttgatcgtc aacaaagata gtccgatcaacaacctgaac gatctgctgg cgaagcggaa 5580 agatctcacc ttcggcaatg gcgatcctaactccacctct ggcttcctcg tccccggtta 5640 ctacgtcttc gccaaaaaca atatctccgccagcgacttc aagcgcaccg tcaacgccgg 5700 gcatgaaacc aacgcgctgg ccgtcgccaacaagcaggtg gatgtggcga ccaacaacac 5760 cgaaaacctc gacaagctga aaacctccgcgccggagaag ctgaaagaac tgaaagtgat 5820 ctggaaatcg ccgctgatcc caggcgatccgatcgtctgg cgtaaaaatc tttccgaaac 5880 caccaaagac aagatctacg acttctttatgaattacggc aaaacgccgg aagagaaagc 5940 ggtgctggaa cgcctgggct gggcgccgttccgcgcctcc agcgacctgc aactggtgcc 6000 gattcgccag ctcgcactgt ttaaagagatgcagggcgtg aaaagcaata aaggactgaa 6060 tgagcaggac aagctggcaa aaaccaccgcgattcaggcg caactggatg acctggaccg 6120 cctgaacaac gcgctaagcg cgatgagttcggtgagtaaa gcggtgcagt aaatcgtagg 6180 tcggataaga cgccccggcg tcgcatccgacaatgtgcag gcgttgatgc cggatgcggt 6240 gcaagcacct tatccggcct acagaccggagccaaacatg caaaccatca ccatcgcccc 6300 acccaagcgc agctggttct cgcttctgagctgggccgtt gttctcgccg tgctggtcgt 6360 ctcgtggcag ggcgcggaaa tggccccgctcacgctgatt aaagacggcg gcaacatggc 6420 aaccttcgct gccgacttct tcccgcccgatttcagccag tggcaggatt acctcaccga 6480 aatggccgtc acgctgcaaa tcgccgtctggggcaccgcg ctggcggtgg ttctctccat 6540 cccctttggc ctgatgagcg ccgaaaacctggtgccgtgg tgggtttacc agcccgttcg 6600 ccgcctgatg gacgcctgcc gcgccattaacgaaatggtc ttcgccatgc tgttcgtggt 6660 cgccgtcggt ctcggaccgt tcgctggcgtgctggcgcta tttatccaca ccaccggcgt 6720 gctctccaag ctgctttccg aagcggtagaagcaattgaa cctggcccgg tggaaggcat 6780 tcgcgccacc ggtgccaaca agctcgaagagatcctctac ggcgtgctgc cgcaggtgat 6840 gccgctgctg atctcctact ccctctatcgcttcgaatcc aacgtccgct cggcgaccgt 6900 cgtcggcatg gtcggcgcgg gcgggatcggcgtcaccctg tgggaagcga ttcgcggttt 6960 ccagttccaa caaacctgcg ccctgatggtgcttatcatc gtcacggtca gcctgctgga 7020 tttcctctct caacggttgc gtaagcactttatctgataa gcgaggcatt gatatctatg 7080 cacttgtcta cacatccgac cagctacccaacacgctatc aagagatagc cgcaaaactt 7140 gagcaggagc ttcgtcaaca ctaccgctgcggcgactatc ttcccgccga gcagcaactg 7200 gcagcgcgct ttgaggtgaa tcgccacaccctgcgccgcg ccatcgacca actggtggaa 7260 aaaggctggg tacagcgccg tcagggcgtcggcgtgctgg tgctgatgcg cccgttcgat 7320 tacccgctca acgcccaggc gcgttttagccagaatctgc tggatcaggg cagccatccc 7380 accagcgaaa aactgctttc ggtattgcgccccgcgtccg gccacgtcgc tgacgcactg 7440 gggattaccg agggggagaa cgtcatccacctgcgcaccc tgcgtcgggt caacggcgtc 7500 gcgctctgtt taatcgacca ctacttcgcggacctcaccc tctggccgac gctgcaacgc 7560 ttcgacagcg gctcgctgca cgattttctgcgcgagcaaa ccggaattgc gctgcgccgc 7620 agccagacgc ggatcagcgc ccgccgcgcccaggccaaag agtgccagcg tcttgaaatc 7680 ccgaatatgt cgccgctgct gtgcgtgcgcacccttaacc accgtgacgg tgaaagcagc 7740 ccggcggagt actccgtcag cctgacgcgcgccgacatga ttgaattcac tatggagcac 7800 tgaatgcacg cagataccgc gacccgccagcactggatgt ccgtgctggc gcacagccaa 7860 ccggctgaac tggcagcacg cctgaacgcgctaaacatca ccgccgacta tgaggtgatc 7920 cgcgccgctg aaactggcct ggtacagattcaggcgcgga tgggcggcac cggcgaacgt 7980 ttttttgccg gcgacgccac gctgacccgcgccgccgtgc gcctgactga cggcacgctc 8040 ggctacagct gggtgctggg gcgtgataaacagcacgccg aacgctgcgc gctgattgac 8100 gcgctgatgc agcaatctcg ccactttcaaaacttatcag aaacccttat tgccccgctg 8160 gacgctgacc gtatggcacg cattgccgcacgccaggccg aagtgaacgc cagccgggtc 8220 gacttcttta cgatggttcg cggagacaacgcatgaccct ggaaaccgct tttatgcttc 8280 ccgtgcagga tgcccagcac agttttcgtcgcctgttaaa ggccatgagc gagccgggcg 8340 tgattgtcgc cctgcatcag ctcaaacgcggctggcaacc gctgaatatc gccaccacca 8400 gcgtgctgct gacgctggcc gataacgacacgccggtgtg gctttctacc ccattaaata 8460 acgatatcgt caaccagagc ctgcgttttcataccaacgc gccgctggtc agccagccgg 8520 aacaggcgac cttcgcggtg acggatgaggcgatttccag cgaacagctc aacgcccttt 8580 ccaccggcac cgccgttgcg ccggaagcgggcgcgacgct gattttacag gtcgccagcc 8640 tgagcggcgg gcgcatgttg cgtctcaccggcgcgggtat tgccgaagaa cgaatgatcg 8700 ctccgcagct gccggagtgc attctgcacgaactcaccga gcgcccgcac ccgttcccgc 8760 tcggcatcga cctgatcctg acctgcggcgaacgcctgct ggctattccg cgaaccacgc 8820 atgtggaggt gtgctgatgt acgttgccgtaaaagggggc gaaaaggcga tcgacgccgc 8880 ccacgccctg caagagagcc gacgccggggcgataccgat ttgcctgaac tgagcgtcgc 8940 ccagattgaa cagcagctta acctcgcggtagatcgcgtg atgaccgaag gcggcattgc 9000 cgaccgcgaa ctggcggcgc tggcgctgaaacaggccagc ggcgataacg ttgaagcgat 9060 tttcctgctg cgcgcctacc gcaccacgttggcgaagctg gcggtaagcg agccgctcga 9120 caccaccggg atgcgtctcg aacgccgtatctccgccgtt tataaagaca ttcccggcgg 9180 ccagctgctt ggcccaacct acgactacacccatcgcctg ctcgatttta ccctgctggc 9240 aaacggcgaa gcgccgacgc tgaccaccgccgacagcgaa caacagccgt cgccgcacgt 9300 tttcagcctg ctggcgcgtc aggggctggcgaagtttgaa gaggatagcg gcgcacagcc 9360 ggatgacatc acccgcacgc cgccggtttacccctgctca cgttcttccc gtttgcagca 9420 gttgatgcgc ggcgacgaag gctatttgctggcgctggcc tactccaccc agcgtggtta 9480 cggacgcaat cacccgttcg cgggcgagatccgcagtggt tacatcgacg tgtcgattgt 9540 gccggaagag ctgggatttg cggtaaacgtcggcgaacta ctgatgaccg agtgtgaaat 9600 ggtcaacggt tttatcgacc cgccggatgagccgccgcac ttcacgcgcg gctacgggct 9660 ggtattcggc atgagcgagc gcaaagcgatggcaatggcg ctggtcgatc gtgcgttgca 9720 ggctccggaa tacggcgagc acgcgacaggcccggcgcag gatgaagagt ttgtgctggc 9780 acatgccgac aacgtcgaag ccgcaggctttgtctcgcac ctcaaactcc cccactacgt 9840 cgatttccag gccgaactgg agctactcaaacgtctgcaa caggagaaga accatggcta 9900 atctgagcgg ctacaacttt gcctacctcgacgagcagac caaacgcatg atccgccgcg 9960 ccatcttaaa agcggtggcg atccccggttatcaggtgcc gtttggcggg cgcgagatgc 10020 cgatgccata cggctgggga accggcggcatacagctcac cgccagcgtg attggcgaaa 10080 gcgacgtgct aaaggtgatt gaccagggtgcggatgacac caccaacgcc gtgtcgattc 10140 gcaacttctt taagcgcgtg accggggtaaacaccactga acgtacggac gatgcgacgc 10200 ttatccagac gcgtcaccgc atccccgaaacgccgctgac cgaagatcag atcattatct 10260 tccaggtgcc aatcccggaa ccgctgcgctttatcgagcc gcgcgaaacg gaaacccgca 10320 ccatgcacgc gctggaagag tacggcgtgatgcaggtgaa actgtatgaa gatatcgccc 10380 gcttcggtca tatcgccact acctacgcctatccggtgaa ggtgaacggg cgctacgtaa 10440 tggacccgtc gccgatcccg aaattcgataacccaaaaat ggacatgatg cccgccctgc 10500 aactgttcgg cgcggggcgc gagaagcgcatctatgcggt gccgccgttt acccgcgtgg 10560 aaagtctcga tttcgacgat cacccgttcaccgttcagca gtgggatgag ccatgcgcca 10620 tctgcggatc gacccacagc tatcttgatgaagtggtgct ggatgacgcc ggaaaccgca 10680 tgtttgtctg ctccgatacc gattattgccgccaacagag cgaggcaaaa aaccaatgaa 10740 tcaaccgtta ctttcggtca ataacctgacccacctttac gcgccgggca aaggctttag 10800 cgatgtctct tttgatttat ggccgggggaagtgctgggc attgtcgggg aatccggctc 10860 cgggaagacc acgctgctga agtcgatctccgcgcgcctg acgccgcagc agggggaaat 10920 tcactacgag aaccgttcgc tgtatgcaatgagcgaggcc gaccgccgtc gcctgctgcg 10980 taccgaatgg ggcgtggtgc atcagcatccactcgacggc ctgcgccgcc aggtgtcggc 11040 aggcggcaat atcggcgagc ggctgatggcgaccggggca cgtcattacg gcgatattcg 11100 tgccaccgcg cagaagtggc tggaagaggtggagattccc gccaaccgga tcgacgacct 11160 gccgaccacc ttttccggcg gtatgcagcagcgtttgcag attgcccgca acctggtgac 11220 gcatccgaag ctggtgttta tggatgaaccgaccggcggg ctggatgtgt cggtgcaggc 11280 ccgcctgctc gacctgctgc gcggcctggtggtggagctg aacctcgcgg tggtgattgt 11340 cacccatgat ttaggcgtcg cccgcctgctggcggaccgt ttgctggtga tgaagcaggg 11400 gcaagtggtg gagagtgggt taaccgaccgcgtgctcgac gacccgcatc atccgtatac 11460 acagctgctg gtgtcatcgg ttttgcagaattgagccggt gccggatgcg gcgtaaacgc 11520 cttatccggc ctacaaatgc gctccccgtaggtcggataa gacgcgtcag cgtcgcatcc 11580 gacacccgaa ccacgaggcg aaaaatgattaacgtacaaa acgtcagtaa aaccttcatc 11640 ctgcaccagc aaaacggcgt gcgcctgcccgtcctcaatc gcgcctcgct caccgtcaac 11700 gcgggcgaat gcgtggtgct ccacggccattccggcagcg gcaaatcaac tctgctacgc 11760 tcgctgtacg ccaactatct acccgacgaaggtcaaatcc agatcaaaca cggtgacgag 11820 tgggtagacc tggtcaccgc gccagcgcgcaaagtggtgg aaatccgcaa aaccaccgtc 11880 ggctgggtga gccagtttct gcgcgtcatcccgcgtatct cagcactgga agtggtgatg 11940 cagccgctgc tcgataccgg cgttccgcgtgaagcctgcg ccgctaaagc cgcgcgtctt 12000 ctcacccgcc tgaacgtgcc ggaacgcctgtggcacctgg caccatcgac attttccggt 12060 ggcgaacagc agcgcgtcaa catcgcccgcggctttatcg tcgactaccc cattctgctg 12120 cttgacgaac ctaccgcctc gctggacgccaaaaacagcg ccgcggtggt ggaactgatt 12180 cgcgaagcca aaacccgtgg cgcagccatcgtaggcatct tccatgacga agctgtacgt 12240 aatgacgtcg ccgaccgcct gcacccaatgggagcctctt catgattatc aataacgtta 12300 agctggtgct ggaaaacgag gtggtaagcggttcgctgga ggtgcagaac ggcgaaatcc 12360 gcgcctttgc cgaaagccag agccgcctgccggaggcgat ggacggcgaa ggcggctggc 12420 tgctgccggg gctgattgag ctgcataccgataatctgga taaattcttc accccgcgcc 12480 cgaaagttga ctggcctgcc cactcggcgatgagcagcca cgacgcgctg atggtggcga 12540 gcggcatcac caccgtactg gatgccgtggcaattggcga cgtgcgcgac ggcggcgatc 12600 ggctggagaa tctggagaag atgatcaacgccatcgaaga gacgcagaaa cgcggcgtca 12660 accgcgccga gcaccgtctg catctgcgctgcgaactgcc gcatcacacc acgctgccgc 12720 tgtttgaaaa actggtgcag cgcgagccggtgacgctggt gtcgctgatg gaccactcgc 12780 cgggccagcg ccagttcgcc aaccgcgagaagtatcgcga atattatcag ggcaaatact 12840 ccctcactga tgcgcagatg cagcagtacgaagaagagca actggcgctc gccgcacgct 12900 ggtcgcagcc gaatcgcgaa tccatcgccgccctgtgccg cgcgcgaaaa attgcgcttg 12960 ccagccacga tgacgccacc cacgcccacgttgctgaatc tcaccagctt ggcagcgtga 13020 tcgccgaatt tcccaccacg ttcgaagcggcggaagcctc gcgcaagcat ggcatgaacg 13080 tgctgatggg cgcgccgaat attgtgcgcggcggctcgca ctccggcaac gtggcggcca 13140 gtgaactggc gcagcttggc ctgctggatatcctctcttc cgactactac cccgccagcc 13200 tgctcgatgc ggcatttcgc gtcgccgatgacgagagcaa ccgctttacg ctgccgcagg 13260 cggtgaagct ggtgactaaa aatccagcgcaggcgcttaa tctccaggat cgcggggtga 13320 ttggcgaggg caaacgcgcc gacctggtgctggcgcatcg caaggacaat catattcata 13380 tcgaccacgt ctggcgtcag ggtaaaagggtgttctgatg atgggaaaac tgatttggtt 13440 aatggggccg tccggctccg ggaaagacagcctgctggcg gaactccgcc tgcgggaaca 13500 aactcagtta ctggtggcgc atcgctacatcacgcgcgat gccagcgccg gaagtgaaaa 13560 ccatatcgcc ctgagcgagc aggagttttttacccgcgcg gggcaaaatc tgttggcctt 13620 aagctggcac gctaacggtc tgtattatggcgtcggcgtc gagattgatc tctggctgca 13680 cgccggattc gacgtgctgg tcaacggctcacgcgcccat ctgccgcagg cgcgggcgcg 13740 ctatcaatcg gcgctgctgc ccgtctgtttacaggtttcg ccggagatcc tccgccagcg 13800 cctggaaaac cgtggccgtg aaaacgccagtgaaattaac gcccgcctgg cgcgcgccgc 13860 ccgctatact ccacaggatt gccatacgctcaacaatgac ggcagcctgc gccagtcggt 13920 cgacacgctg ctgacgctga tccatcagaaggagaaacac catgcctgct tgtgagcttc 13980 gcccggccac gcagtacgac accgacgcggtttacgcgct gatttgtgag ctaaaacagg 14040 cggagtttga ccaccacgcg tttcgcgtgggttttaacgc caatctgcgc gacccaaaca 14100 tgcgctacca tctggcgctg cttgatggcgaagttgtcgg catgatcggc ctgcatttgc 14160 agtttcatct gcatcatgtc aactggatcggcgaaattca ggagttggtg gtaatgccgc 14220 aggcgcgcgg tctgaacgtc ggcagtaagttactggcgtg ggcagaagaa gaagcccgcc 14280 aggccggggc cgaaatgacc gaactttcgaccaacgtgaa gcgccacgac gcgcaccgtt 14340 tctatctgcg cgaaggctac gagcagagccacttccgctt caccaaggcg ctgtaacatg 14400 agcctgaccc tcacgctcac cggcaccggcggcgcacagg gcgttccggc atggggctgc 14460 gagtgtgcgg cctgcgccag agcgcggcgctcgccgcagt atcgccgcca accgtgcagc 14520 ggcgtagtga agtttaacga cgcaatcaccctgatcgacg ccgggctgca cgatctcgcc 14580 gatcgctggt cgcccggatc gttccagcagtttttgctga cgcattatca tatggatcac 14640 gtccaggggc tgtttccgct gcgctggggcgttggcgatc cgatcccggt ttacggcccg 14700 ccggatgaac agggctgcga cgatctgtttaaacatccgg gcctgcttga tttcagccac 14760 acggtggaac cgtttgtggt gtttgatttgcaggggttac aggtcacgcc cctgccgctc 14820 aaccactcaa aactgacctt cggttatctgctggaaacgg cacacagccg ggtggcgtgg 14880 ctgtctgaca ccgcaggctt gccggaaaaaacgctgaaat ttttacgcaa taatcagccg 14940 caggtaatgg tgatggattg cagtcacccgccgcgcgcgg atgcaccgcg taatcactgt 15000 gatttaaata ccgtgcttgc gctgaatcaggttatccgct cgccacgggt gattctgacc 15060 catatcagcc accagtttga tgcgtggctgatggaaaacg cactaccgtc agggtttgag 15120 gtggggtttg atgggatgga gattggggtggcgtgatgag agggaatgtg cgcgctggcc 15180 ccctcaccct aaccctctcc ccagaggggcgaggggaccg attgtgctcg atattgaata 15240 ttgcgctcgt tttctccctc tccccattggggtgaggggc gatgcctgct ccatacccaa 15300 cctcatcgcc catactcatc ttccattctccgctcttcat cctccagttg ccgacgctcc 15360 tgatcaagct ggcgctggcg atcgtccagctgcctgcggc gatcttcaaa ctggcggcgg 15420 cggtcgtcat attgtctgcg ccgatcgtcgctcacttcac gctgccagcc gtggtcgcgc 15480 gaatcttcat agttgaagcg gcgcacgaaaaacgcgaaag cgtttcacga taaatgcgaa 15540 aactttagct ttcgcgcttc aaatgaaacagatgtattaa ttactgcttt ttattcatta 15600 catggggatc c 15611 2 11672 DNAEscherichia coli 2 gaattcccgt taactcttca tctgttagtc acttttaattaaccaaatcg tcacaataat 60 ccgccacgat ggagccactt ttttagggag gctgcatcatgcaaacgatt atccgtgtcg 120 agaagctcgc caaaaccttc aatcagcatc aggcgctgcatgcggttgat ctgaacattc 180 atcacggtga aatggtggct ctgcttgggc cgtcgggttccggaaaatcc acccttttac 240 gtcacttaag cggtttgatt accggcgata aatctgtcggtagccatatc gagctgctgg 300 gccgcacagt ccagcgcgaa ggccgcctgg cccgcgatatccgcaaaagc cgcgcccata 360 ccggctacat attccaacaa ttcaacctgg tgaaccgcctgagcgtactg gagaacgtgc 420 tgattggcgc gctcggcagc acgccgttct ggcgcacctgttttagctgg ttcaccggcg 480 agcagaaaca gcgcgcgtta caggcgctga cccgcgttggcatggtgcat tttgcccatc 540 agcgcgtttc caccctctcc ggcggccagc agcaacgtgtggcgattgcc cgtgcgctga 600 tgcagcaggc gaaagtgatt ctggccgatg aacccatcgcctcgctggac ccagaatcag 660 cgcgcatcgt gatggacacc ctgcgcgaca tcaaccagaacgacggcatc accgtggtcg 720 tcacgctgca tcaggtggat tacgccctgc gctactgcgaacgcatcgtc gccctgcgcc 780 aggggcacgt cttctacgac ggcagcagcc aacagtttgataacgaacgt tttgaccatc 840 tctaccgcag cattaaccgc gtcgaagaga acgcgaaagctgcctgacat ccccatcatt 900 gaggaaaacg aatgaacgct aagataattg cctcgctggccttcaccagc atgttcagcc 960 tcagcaccct gttaagcccg gcgcacgccg aagagcaggaaaaggcgttg aatttcggca 1020 ttatttcaac ggaatcacag caaaacctga aaccgcaatggacgccgttc ttgcaggata 1080 tggagaagaa gctgggcgtg aaggtcaacg ccttctttgccccggactac gcgggcatta 1140 tccaggggat gcgcttcaat aaagtggata tcgcctggtacggcaatctg tcggcgatgg 1200 aagcggtgga tcgcgccaat ggccaggtct tcgcccagacggtcgcggcg gatggatcgc 1260 cgggttactg gagcgtgttg atcgtcaaca aagacagtccgatcaacaac ctgaacgatc 1320 tgctggcgaa gcggaaagat ctcacctttg gcaatggcgatcctaactcc acctctggct 1380 tcctcgtccc cggctactac gtcttcgcca aaaacaatatctccgccagc gacttcaagc 1440 gcaccgtcaa cgccgggcat gaaaccaacg cgctggccgtcgccaacaag caggtggatg 1500 ttgccaccaa caacaccgaa aacctcgaca agctgaaaacctccgcgcca gagaagctga 1560 aagaactgaa ggtgatctgg aagtcgccgc tgatcccaggcgatccgatc gtctggcgca 1620 agaatctttc cgaaaccacc aaagacaaga tctacgacttctttatgaac tacggcaaaa 1680 cgccggaaga aaaagcggtg ctggaacgcc tgggctgggcgccattccgc gcttccagcg 1740 acctgcaact ggtgccgatt cgccagctcg cgctgtttaaagagatgcag ggcgtgaaaa 1800 gcaataaagg actgaatgag caggacaagc tggcaaaaaccaccgagatt caggcgcagc 1860 tggatgacct ggaccgcctg aacaacgcgc taagcgcgatgagttcggtg agtaaagcgg 1920 tgcagtaaat cgtaggtcgg ataagacgcc ccggcgtcgcatccgacaat gtgcaggcgt 1980 tgatgccgga tgcggtgcaa gcaccttatc cggcctacagaccggagcca aacatgcaaa 2040 ccatcaccat cgccccaccc aagcgcagct ggttctcgcttctgagctgg gccgttgtac 2100 tcgccgtgtt ggtcgtctcg tggcagggcg cggaaatggccccgcttacg ctgatcaaag 2160 acggcggcaa catggcgacg ttcgccgccg acttcttcccgcccgatttc agccagtggc 2220 aggattacct caccgaaatg gccgtcacgc tgcaaatcgccgtctggggc accgcgctgg 2280 cggtggttct ctccatcccc tttggcctga tgagcgccgaaaacctggtg ccgtggtggg 2340 tttaccagcc cgttcgccgc ctgatggacg cctgccgcgccattaacgaa atggtcttcg 2400 ccatgctgtt cgtggtcgcc gtcggcctcg gcccgttcgctggcgtgctg gcgtgctggc 2460 gctgtttatc cacaccaccg gcgtgctctc caagctgctttccgaagcgg tggaagcgat 2520 tgagcccggc ccggtggaag gcattcgcgc caccggtgccaacaagctcg aagagatcct 2580 ctacggcgtg ctgccacagg tgatgccact gctgatctcctactccctct atcgcttcga 2640 atccaacgtc cgctcggcga ccgtcgtcgg catggtcggcgcaggcggga tcggcgtcac 2700 cctgtgggaa gcgattcgcg gtttccagtt ccaacaaacctgcgccctga tggtgcttat 2760 catcgtcacg gtcagcctgc tggatttcct ctctcaacggttgcgtaagc actttatctg 2820 ataagcgagg cattgatatc tatgcacttg tctacacatccgaccagcta cccaacacgc 2880 tatcaagaga tagccgcaaa acttgagcag gagcttcgtcaacactaccg ctgcggcgac 2940 tatcttcccg ccgagcagca actggcagcg cgctttgaggtgaatcgcca caccctgcgc 3000 cgcgccatcg accaactggt ggaaaaaggc tgggtacagcgccgtcaggg cgtcggcgtg 3060 ctggtgctga tgcgcccgtt cgattacccg ctcaacgcccaggcgcgttt tagccagaat 3120 ctgctggatc agggcagcca tcccaccagc gaaaaactgctttcggtatt gcgccccgcg 3180 tccggccacg tcgctgacgc actggggatt accgagggggagaacgtcat ccacctgcgc 3240 accctgcgtc gtgtcaacgg cgtcgcgctc tgtttaatcgaccactactt cgcggacctc 3300 accctctggc cgacgctgca acgcttcgac agcggctcgctgcacgattt tctgcgcgag 3360 caaaccggaa ttgcgctgcg ccgcagccag acgcggatcagcgcccgccg cgcccaggcc 3420 aaagagtgcc agcgtcttga aatcccgaat atgtcgccgctgctgtgcgt gcgcaccctt 3480 aaccaccgtg acggtgaaag cagcccggcg gagtactccgtcagcctgac gcgcgccgac 3540 atgattgaat tcactatgga gcactgaatg cacgcagataccgcgacccg ccagcactgg 3600 atgtccgtgc tggcgcacag ccaaccggct gaactggcagcacgcctgaa cgcgctaaac 3660 atcaccgccg actatgaggt gatccgcgcc gctgaaactggcctggtaca gattcaggcg 3720 cggatgggcg gcaccggcga acgttttttt gccggcgacgccacgctgac ccgcgccgcc 3780 gtgcgcctga ctgacggcac gctcggctac agctgggtgcaggggcgtga taaacagcac 3840 gccgaacgct gcgcgctgat tgacgcgctg atgcagcaatctcgccactt tcaaaactta 3900 tcagaaaccc ttattgcccc gctggacgct gaccgtatggcacgcattgc cgcacgccag 3960 gccgaagtga acgccagccg ggtcgacttc tttacgatggttcgcggaga caacgcatga 4020 ccctggaaac cgcttttatg cttcccgtgc aggatgcccagcacagtttt cgtcgcctgt 4080 taaaggccat gagcgagccg ggcgtgattg tcgccctgcatcagctcaaa cgcggctggc 4140 aaccgctgaa tatcgccacc accagcgtgc tgctgacgctggccgataac gacacgccgg 4200 tgtggctttc taccccatta aataacgata tcgtcaaccagagcctgcgt tttcatacca 4260 acgcgccgct ggtcagccag ccggaacagg cgaccttcgcggtgacggat gaggcgattt 4320 ccagcgaaca gctcaacgcc ctttccaccg gcaccgccgttgcgccggaa gcgggtgcga 4380 cgctgatttt acaggtcgcc agcctgagcg gcggacgcatgttgcgcctt actggtgcgg 4440 gtattgccga agaacgaatg atcgctccgc agctgccggagtgcattctg cacgaactca 4500 ccgagcgccc gcatccgttc ccgctcggca tcgacctgatcctgacctgt ggcgagcgcc 4560 tgctggctat tccgcgaacc actcatgtgg aggtgtgctgatgtacgttg ccgtgaaagg 4620 gggcgagaag gcgatcgacg ccgcccacgc cctgcaagagagccgacgcc gaggcgatac 4680 cgatttgccc gaactgagcg tcgcccagat tgaacagcagcttaacctcg cggtagatcg 4740 cgtgatgacc gaaggcggca ttgccgaccg cgaactggcggcgctggcgc tgaaacaggc 4800 cagcggcgat aacgttgaag cgattttcct gctgcgcgcctaccgcacca cgttggcgaa 4860 gctggcggta agcgagccgc tcgacaccac cgggatgcgtctcgaacgcc gtatctccgc 4920 cgtttataaa gacattcccg gcggccagct gcttggcccaacctacgact acacccatcg 4980 cctgctcgat tttaccctgc tggcaaacgg cgaagcgccgacgctgacca ccgccgacag 5040 cgaacagcag ccgtcgccgc acgttttcag cctgctggcgcgtcaggggc tggcgaagtt 5100 tgaagaggat agcggcgcac agccggatga catcacccgcacgccgccgg tttacccctg 5160 ctcacgctcc tcccgtttgc agcagttgat gcgcggcgacgaaggctatt tgctggcgct 5220 ggcctactcc acccaacgcg gttacgggcg caatcacccgttcgcaggcg agatccgcag 5280 cggctatatc gacgtgtcga ttgtgccgga agagctgggatttgcggtga acgtcggcga 5340 actgctgatg actgagtgtg aaatggttaa cggttttatcgacccgccgg gtgagccgcc 5400 gcacttcacg cgcggctacg ggctggtgtt cggcatgagcgagcgcaaag cgatggcgat 5460 ggcgctggtc gaccgcgctc tgcaagcccc ggagtacggcgagcacgcga caggcccggc 5520 gcaggatgaa gagttcgtgc tggcacatgc cgacaacgtcgaagccgcag gctttgtctc 5580 acacctcaaa ctcccccact acgtcgattt ccaggccgaactggagctac tcaaacgtct 5640 gcaacaggag cagaaccatg gctaatctga gcggctacaactttgcctac ctcgacgagc 5700 agaccaaacg catgatccgc cgcgccatct taaaagcggtggcgatcccc ggttatcagg 5760 tgccgtttgg cgggcgcgag atgccgatgc cgtacggctggggaaccggc ggcattcagc 5820 ttaccgccag cgtgattggc gaaagcgacg tgctgaaggtgattgaccag ggcgcggatg 5880 acaccaccaa cgccgtgtcg attcgcaact tcttcaagcgcgtgaccggg gtaaacacca 5940 cggaacgtac ggacgatgcg acggttatcc agacgcgtcaccgcatcccc gaaacgccgc 6000 tgaccgaaga tcagataatt atcttccagg tgccaatccccgagccgctg cgctttatcg 6060 agccgcgcga aacggaaacc cgcaccatgc acgcgctggaagagtacggc gtgatgcagg 6120 tgaaactgta tgaagatatc gcccgcttcg gtcatatcgccaccacctac gcctatccgg 6180 tgaaggtaaa tgggcgctac gtgatggacc cgtcgccgatcccgaaattc gataacccaa 6240 aaatggacat gatgcccgcc ctgcaactgt tcggcgcggggcgcgagaag cgcatctatg 6300 cggtgccgcc gtttacccgc gtggaaagtc tcgatttcgacgatcacccg ttcaccgttc 6360 agcagtggga tgagccatgc gccatctgcg gatcgacccacagctatctt gatgaagtgg 6420 tgctggatga cgccggaaac cgcatgtttg tctgctccgataccgattat tgccgccaac 6480 agagcgaggc aaaaaaccaa tgaatcaacc gttactttcggtcaataacc tgacccacct 6540 ttacgcgccg ggcaaaggct ttagcgatgt ctcttttgatttatggccgg gggaagtgct 6600 gggcattgtc ggggaatccg gctccgggaa gaccacgctgctgaagtcga tctccgcgcg 6660 cctgacgccg cagcaggggg aaattcacta cgagaaccgttcgctgtatg caatgagcga 6720 ggccgaccgc cgtcgcctgc tgcgtaccga atggggcgtggtgcatcagc atccactcga 6780 cggcctgcgc cgccaggtgt cggcaggcgg caatatcggcgagcggctga tggcgaccgg 6840 ggcacgtcat tacggcgata ttcgtgccac cgcgcagaagtggctggaag aggtggagat 6900 tcccgccaac cggatcgacg acctgccgac caccttttccggcggtatgc agcagcgttt 6960 gcagattgcc cgcaacctgg tgacgcatcc gaagctggtgtttatggatg aaccgaccgg 7020 cgggctggat gtgtcggtgc aggcccgcct gctcgacctgctgcgcggcc tggtggtgga 7080 gctgaacctc gcggtggtga ttgtcaccca tgatttaggcgtcgcccgcc tgctggcgga 7140 ccgtttgctg gtgatgaagc aggggcaagt ggtggagagtgggttaaccg accgcgtgct 7200 cgacgacccg catcatccgt atacacagct gctggtgtcatcggttttgc agaattgagc 7260 cggtgccgga tgcggcgtaa acgccttatc cggcctacaaatgcgctccc cgtaggtcgg 7320 ataagacgcg tcagcgtcgc atccgacacc cgaaccacgaggcgaaaaat gattaacgta 7380 caaaacgtca gtaaaacctt catcctgcac cagcaaaacggcgtgcgcct gcccgtcctc 7440 aatcgcgcct cgctcaccgt caacgcgggc gaatgcgtggtgctccacgg ccattccggc 7500 agcggcaaat caactctgct acgctcgctg tacgccaactatctgcccga cgaaggtcaa 7560 atccagatca aacacggtga cgagtgggta gacctggtcaccgcgccagc gcgcaaagtg 7620 gtggaaatcc gcaaaaccac cgtcggctgg gtgagccagtttctgcgcgt catcccgcgt 7680 atctcagcac tggaagtggt gatgcagccg ctgctcgataccggcgttcc gcgtgaagcc 7740 tgcgccgcta aagccgcgcg tcttctcacc cgcctgaacgtgccggaacg cctgtggcac 7800 ctggcaccat cgacattttc cggtggcgaa cagcagcgcgtcaacatcgc ccgcggcttt 7860 atcgtcgact accccattct gctgcttgac gaacctaccgcctcgctgga cgccaaaaac 7920 agcgccgcgg tggtggaact gattcgcgaa gccaaaacccgtggcgcagc catcgtaggc 7980 atcttccatg acgaagctgt acgtaatgac gtcgccgaccgcctgcaccc aatgggagcc 8040 tcttcatgat tatcaataac gttaagctgg tgctggaaaacgaggtggta agcggttcgc 8100 tggaggtgca gaacggcgaa atccgcgcct ttgccgaaagccagagccgc ctgccggagg 8160 cgatggacgg cgaaggcggc tggctgctgc cggggctgattgagctgcat accgataatc 8220 tggataaatt cttcaccccg cgcccgaaag ttgactggcctgcccactcg gcgatgagca 8280 gccacgacgc gctgatggtg gcgagcggca tcaccaccgtactggatgcc gtggcaattg 8340 gcgacgtgcg cgacggcggc gatcggctgg agaatctggagaagatgatc aacgccatcg 8400 aagagacgca gaaacgcggc gtcaaccgcg ccgagcaccgtctgcatctg cgctgcgaac 8460 tgccgcatca caccacgctg ccgctgtttg aaaaactggtgcagcgcgag ccggtgacgc 8520 tggtgtcgct gatggaccac tcgccgggcc agcgccagttcgccaaccgc gagaagtatc 8580 gcgaatatta tcagggcaaa tactccctca ctgatgcgcagatgcagcag tacgaagaag 8640 agcaactggc gctcgccgca cgctggtcgc agccgaatcgcgaatccatc gccgccctgt 8700 gccgcgcgcg aaaaattgcg cttgccagcc acgatgacgccacccacgcc cacgttgctg 8760 aatctcacca gcttggcagc gtgatcgccg aatttcccaccacgttcgaa gcggcggaag 8820 cctcgcgcaa gcatggcatg aacgtgctga tgggcgcgccgaatattgtg cgcggcggct 8880 cgcactccgg caacgtggcg gccagtgaac tggcgcagcttggcctgctg gatatcctct 8940 cttccgacta ctaccccgcc agcctgctcg atgcggcatttcgcgtcgcc gatgaccaga 9000 gcaaccgctt tacgctgccg caggcggtga agctggtgactaaaaatcca gcgcaggcgc 9060 ttaatctcca ggatcgcggg gtgattggcg agggcaaacgcgccgacctg gtgctggcgc 9120 atcgcaagga caatcatatt catatcgacc acgtctggcgtcagggtaaa agggtgttct 9180 gatgatggga aaactgattt ggttaatggg gccgtccggctccgggaaag acagcctgct 9240 ggcggaactc cgcctgcggg aacaaactca gttactggtggcgcatcgct acatcacgcg 9300 cgatgccagc gccggaagtg aaaaccatat cgccctgagcgagcaggagt tttttacccg 9360 cgcggggcaa aatctgttgg ccttaagctg gcacgctaacggtctgtatt atggcgtcgg 9420 cgtcgagatt gatctctggc tgcacgccgg attcgacgtgctggtcaacg gctcacgcgc 9480 ccatctgccg caggcgcggg cgcgctatca atcggcgctgctgcccgtct gtttacaggt 9540 ttcgccggag atcctccgcc agcgcctgga aaaccgtggccgtgaaaacg ccagtgaaat 9600 taacgcccgc ctggcgcgcg ccgcccgcta tactccacaggattgccata cgctcaacaa 9660 tgacggcagc ctgcgccagt cggtcgacac gctgctgacgctgatccatc agaaggagaa 9720 acaccatgcc tgcttgtgag cttcgcccgg ccacgcagtacgacaccgac gcggtttacg 9780 cgctgatttg tgagctaaaa caggcggagt ttgaccaccacgcgtttcgc gtgggtttta 9840 acgccaatct gcgcgaccca aacatgcgct accatctggcgctgcttgat ggcgaagttg 9900 tcggcatgat cggcctgcat ttgcagtttc atctgcatcatgtcaactgg atcggcgaaa 9960 ttcaggagtt ggtggtaatg ccgcaggcgc gcggtctgaacgtcggcagt aagttactgg 10020 cgtgggcaga agaagaagcc cgccaggccg gggccgaaatgaccgaactt tcgaccaacg 10080 tgaagcgcca cgacgcgcac cgtttctatc tgcgcgaaggctacgagcag agccacttcc 10140 gcttcaccaa ggcgctgtaa catgagcctg accctcacgctcaccggcac cggcggcgca 10200 cagggcgttc cggcatgggg ctgcgagtgt gcggcctgcgccagagcgcg gcgctcgccg 10260 cagtatcgcc gccaaccgtg cagcggcgta gtgaagtttaacgacgcaat caccctgatc 10320 gacgccgggc tgcacgatct cgccgatcgc tggtcgcccggatcgttcca gcagtttttg 10380 ctgacgcatt atcatatgga tcacgtccag gggctgtttccgctgcgctg gggcgttggc 10440 gatccgatcc cggtttacgg cccgccggat gaacagggctgcgacgatct gtttaaacat 10500 ccgggcctgc ttgatttcag ccacacggtg gaaccgtttgtggtgtttga tttgcagggg 10560 ttacaggtca cgcccctgcc gctcaaccac tcaaaactgaccttcggtta tctgctggaa 10620 acggcacaca gccgggtggc gtggctgtct gacaccgcaggtttgccgga aaaaacgctg 10680 aaatttttac gcaataatca gccgcaggta atggtgatggattgcagtca cccgccgcgc 10740 gcggatgcac cgcgtaatca ctgtgattta aataccgtgcttgcgctgaa tcaggttatc 10800 cgctcgccac gggtgattct gacccatatc agccaccagtttgatgcgtg gctgatggaa 10860 aacgcactac cgtcagggtt tgaggtgggg tttgatgggatggagattgg ggtggcgtga 10920 tgagagggaa tgtgcgcgct ggccccctca ccctaaccctctccccagag gggcgagggg 10980 accgattgtg ctcgatattg aatattgcgc tcgttttctccctctcccca ttggggtgag 11040 gggcgatgcc tgctccatac ccaacctcat cgcccatactcatcttccat tctccgctct 11100 tcatcctcca gttgccgacg ctcctgatca agctggcgctggcgatcgtc cagctgcctg 11160 cggcgatctt caaactggcg gcggcggtcg tcatattgtctgcgccgatc gtcgctcact 11220 tcacgctgcc agccgtcgtc gcgcgaatct tcatagtctcgcccacggtc agggttataa 11280 gcgtcattaa tcgcctgctg aatattgcca atggtgtcgtcgataatatc ggcctgggcc 11340 ggaacgtgga cagcgtgagc agggtgaata aaagaaatagcggaaagcgt ttcattagcc 11400 aacctcaaaa agaaactcta tccacattaa tcattactcatccatgcaag tagtggatga 11460 atctcaattt ctccgctgct ctattgccgt aatcgcctccacgcgttgtt gatgacgacc 11520 gccttcgtac tgtgcgccca gccacgcatc cacaatcatttttgccagtt cgaggccaac 11580 cactcgtgaa ccaaaagcca gcacgttggt gtcgttatgctgccgcgaaa gttgcgcgga 11640 ataaggttcg ctacagacga ccgcgcgaat tc 11672 3435 DNA Escherichia coli 3 atgcctgctt gtgagcttcg cccggccacg cagtacgacaccgacgcggt ttacgcgctg 60 atttgtgagc taaaacaggc ggagtttgac caccacgcgtttcgcgtggg ttttaacgcc 120 aatctgcgcg acccaaacat gcgctaccat ctggcgctgcttgatggcga agttgtcggc 180 atgatcggcc tgcatttgca gtttcatctg catcatgtcaactggatcgg cgaaattcag 240 gagttggtgg taatgccgca ggcgcgcggt ctgaacgtcggcagtaagtt actggcgtgg 300 gcagaagaag aagcccgcca ggccggggcc gaaatgaccgaactttcgac caacgtgaag 360 cgccacgacg cgcaccgttt ctatctgcgc gaaggctacgagcagagcca cttccgcttc 420 accaaggcgc tgtaa 435 4 144 PRT Escherichiacoli 4 Met Pro Ala Cys Glu Leu Arg Pro Ala Thr Gln Tyr Asp Thr Asp Ala 15 10 15 Val Tyr Ala Leu Ile Cys Glu Leu Lys Gln Ala Glu Phe Asp His His20 25 30 Ala Phe Arg Val Gly Phe Asn Ala Asn Leu Arg Asp Pro Asn Met Arg35 40 45 Tyr His Leu Ala Leu Leu Asp Gly Glu Val Val Gly Met Ile Gly Leu50 55 60 His Leu Gln Phe His Leu His His Val Asn Trp Ile Gly Glu Ile Gln65 70 75 80 Glu Leu Val Val Met Pro Gln Ala Arg Gly Leu Asn Val Gly SerLys 85 90 95 Leu Leu Ala Trp Ala Glu Glu Glu Ala Arg Gln Ala Gly Ala GluMet 100 105 110 Thr Glu Leu Ser Thr Asn Val Lys Arg His Asp Ala His ArgPhe Tyr 115 120 125 Leu Arg Glu Gly Tyr Glu Gln Ser His Phe Arg Phe ThrLys Ala Leu 130 135 140 5 20 DNA Artificial Sequence Description ofArtificial Sequencesynthetic oligonucleotide 5 aaacaccatg gctgcttgtg 206 35 DNA Artificial Sequence Description of Artificial Sequencesyntheticoligonucleotide 6 gtgacgaatt cgagctcatt acagcgcctt ggtga 35 7 435 DNAArtificial Sequence Description of Artificial Sequencenon-naturallyoccurring nucleotide sequence encoding modified PhnO protein P2A; g-c atnucleotide position 4 7 atg gct gct tgt gag ctt cgc ccg gcc acg cag tacgac acc gac gcg 48 Met Ala Ala Cys Glu Leu Arg Pro Ala Thr Gln Tyr AspThr Asp Ala 1 5 10 15 gtt tac gcg ctg att tgt gag cta aaa cag gcg gagttt gac cac cac 96 Val Tyr Ala Leu Ile Cys Glu Leu Lys Gln Ala Glu PheAsp His His 20 25 30 gcg ttt cgc gtg ggt ttt aac gcc aat ctg cgc gac ccaaac atg cgc 144 Ala Phe Arg Val Gly Phe Asn Ala Asn Leu Arg Asp Pro AsnMet Arg 35 40 45 tac cat ctg gcg ctg ctt gat ggc gaa gtt gtc ggc atg atcggc ctg 192 Tyr His Leu Ala Leu Leu Asp Gly Glu Val Val Gly Met Ile GlyLeu 50 55 60 cat ttg cag ttt cat ctg cat cat gtc aac tgg atc ggc gaa attcag 240 His Leu Gln Phe His Leu His His Val Asn Trp Ile Gly Glu Ile Gln65 70 75 80 gag ttg gtg gta atg ccg cag gcg cgc ggt ctg aac gtc ggc agtaag 288 Glu Leu Val Val Met Pro Gln Ala Arg Gly Leu Asn Val Gly Ser Lys85 90 95 tta ctg gcg tgg gca gaa gaa gaa gcc cgc cag gcc ggg gcc gaa atg336 Leu Leu Ala Trp Ala Glu Glu Glu Ala Arg Gln Ala Gly Ala Glu Met 100105 110 acc gaa ctt tcg acc aac gtg aag cgc cac gac gcg cac cgt ttc tat384 Thr Glu Leu Ser Thr Asn Val Lys Arg His Asp Ala His Arg Phe Tyr 115120 125 ctg cgc gaa ggc tac gag cag agc cac ttc cgc ttc acc aag gcg ctg432 Leu Arg Glu Gly Tyr Glu Gln Ser His Phe Arg Phe Thr Lys Ala Leu 130135 140 taa 435 8 144 PRT Artificial Sequence 8 Met Ala Ala Cys Glu LeuArg Pro Ala Thr Gln Tyr Asp Thr Asp Ala 1 5 10 15 Val Tyr Ala Leu IleCys Glu Leu Lys Gln Ala Glu Phe Asp His His 20 25 30 Ala Phe Arg Val GlyPhe Asn Ala Asn Leu Arg Asp Pro Asn Met Arg 35 40 45 Tyr His Leu Ala LeuLeu Asp Gly Glu Val Val Gly Met Ile Gly Leu 50 55 60 His Leu Gln Phe HisLeu His His Val Asn Trp Ile Gly Glu Ile Gln 65 70 75 80 Glu Leu Val ValMet Pro Gln Ala Arg Gly Leu Asn Val Gly Ser Lys 85 90 95 Leu Leu Ala TrpAla Glu Glu Glu Ala Arg Gln Ala Gly Ala Glu Met 100 105 110 Thr Glu LeuSer Thr Asn Val Lys Arg His Asp Ala His Arg Phe Tyr 115 120 125 Leu ArgGlu Gly Tyr Glu Gln Ser His Phe Arg Phe Thr Lys Ala Leu 130 135 140 9264 DNA Artificial Sequence Description of Artificial Sequence transitpeptide coding sequence 9 atg gct tcc tct atg ctc tct tcc gct act atggtt gcc tct ccg gct 48 Met Ala Ser Ser Met Leu Ser Ser Ala Thr Met ValAla Ser Pro Ala 1 5 10 15 cag gcc act atg gtc gct cct ttc aac gga cttaag tcc tcc gct gcc 96 Gln Ala Thr Met Val Ala Pro Phe Asn Gly Leu LysSer Ser Ala Ala 20 25 30 ttc cca gcc acc cgc aag gct aac aac gac att acttcc atc aca agc 144 Phe Pro Ala Thr Arg Lys Ala Asn Asn Asp Ile Thr SerIle Thr Ser 35 40 45 aac ggc gga aga gtt aac tgc atg cag gtg tgg cct ccgatt gga aag 192 Asn Gly Gly Arg Val Asn Cys Met Gln Val Trp Pro Pro IleGly Lys 50 55 60 aag aag ttt gag act ctc tct tac ctt cct gac ctt acc gattcc ggt 240 Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Asp Leu Thr Asp SerGly 65 70 75 80 ggt cgc gtc aac tgc atg cag gcc 264 Gly Arg Val Asn CysMet Gln Ala 85 10 88 PRT Artificial Sequence 10 Met Ala Ser Ser Met LeuSer Ser Ala Thr Met Val Ala Ser Pro Ala 1 5 10 15 Gln Ala Thr Met ValAla Pro Phe Asn Gly Leu Lys Ser Ser Ala Ala 20 25 30 Phe Pro Ala Thr ArgLys Ala Asn Asn Asp Ile Thr Ser Ile Thr Ser 35 40 45 Asn Gly Gly Arg ValAsn Cys Met Gln Val Trp Pro Pro Ile Gly Lys 50 55 60 Lys Lys Phe Glu ThrLeu Ser Tyr Leu Pro Asp Leu Thr Asp Ser Gly 65 70 75 80 Gly Arg Val AsnCys Met Gln Ala 85 11 696 DNA Artificial Sequence Description ofArtificial Sequence CTP-AMPA acetyltransferase coding sequence and aminoacid sequence translation 11 atg gct tcc tct atg ctc tct tcc gct act atggtt gcc tct ccg gct 48 Met Ala Ser Ser Met Leu Ser Ser Ala Thr Met ValAla Ser Pro Ala 1 5 10 15 cag gcc act atg gtc gct cct ttc aac gga cttaag tcc tcc gct gcc 96 Gln Ala Thr Met Val Ala Pro Phe Asn Gly Leu LysSer Ser Ala Ala 20 25 30 ttc cca gcc acc cgc aag gct aac aac gac att acttcc atc aca agc 144 Phe Pro Ala Thr Arg Lys Ala Asn Asn Asp Ile Thr SerIle Thr Ser 35 40 45 aac ggc gga aga gtt aac tgc atg cag gtg tgg cct ccgatt gga aag 192 Asn Gly Gly Arg Val Asn Cys Met Gln Val Trp Pro Pro IleGly Lys 50 55 60 aag aag ttt gag act ctc tct tac ctt cct gac ctt acc gattcc ggt 240 Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Asp Leu Thr Asp SerGly 65 70 75 80 ggt cgc gtc aac tgc atg cag gcc atg gct gct tgt gag cttcgc ccg 288 Gly Arg Val Asn Cys Met Gln Ala Met Ala Ala Cys Glu Leu ArgPro 85 90 95 gcc acg cag tac gac acc gac gcg gtt tac gcg ctg att tgt gagcta 336 Ala Thr Gln Tyr Asp Thr Asp Ala Val Tyr Ala Leu Ile Cys Glu Leu100 105 110 aaa cag gcg gag ttt gac cac cac gcg ttt cgc gtg ggt ttt aacgcc 384 Lys Gln Ala Glu Phe Asp His His Ala Phe Arg Val Gly Phe Asn Ala115 120 125 aat ctg cgc gac cca aac atg cgc tac cat ctg gcg ctg ctt gatggc 432 Asn Leu Arg Asp Pro Asn Met Arg Tyr His Leu Ala Leu Leu Asp Gly130 135 140 gaa gtt gtc ggc atg atc ggc ctg cat ttg cag ttt cat ctg catcat 480 Glu Val Val Gly Met Ile Gly Leu His Leu Gln Phe His Leu His His145 150 155 160 gtc aac tgg atc ggc gaa att cag gag ttg gtg gta atg ccgcag gcg 528 Val Asn Trp Ile Gly Glu Ile Gln Glu Leu Val Val Met Pro GlnAla 165 170 175 cgc ggt ctg aac gtc ggc agt aag tta ctg gcg tgg gca gaagaa gaa 576 Arg Gly Leu Asn Val Gly Ser Lys Leu Leu Ala Trp Ala Glu GluGlu 180 185 190 gcc cgc cag gcc ggg gcc gaa atg acc gaa ctt tcg acc aacgtg aag 624 Ala Arg Gln Ala Gly Ala Glu Met Thr Glu Leu Ser Thr Asn ValLys 195 200 205 cgc cac gac gcg cac cgt ttc tat ctg cgc gaa ggc tac gagcag agc 672 Arg His Asp Ala His Arg Phe Tyr Leu Arg Glu Gly Tyr Glu GlnSer 210 215 220 cac ttc cgc ttc acc aag gcg ctg 696 His Phe Arg Phe ThrLys Ala Leu 225 230 12 232 PRT Artificial Sequence 12 Met Ala Ser SerMet Leu Ser Ser Ala Thr Met Val Ala Ser Pro Ala 1 5 10 15 Gln Ala ThrMet Val Ala Pro Phe Asn Gly Leu Lys Ser Ser Ala Ala 20 25 30 Phe Pro AlaThr Arg Lys Ala Asn Asn Asp Ile Thr Ser Ile Thr Ser 35 40 45 Asn Gly GlyArg Val Asn Cys Met Gln Val Trp Pro Pro Ile Gly Lys 50 55 60 Lys Lys PheGlu Thr Leu Ser Tyr Leu Pro Asp Leu Thr Asp Ser Gly 65 70 75 80 Gly ArgVal Asn Cys Met Gln Ala Met Ala Ala Cys Glu Leu Arg Pro 85 90 95 Ala ThrGln Tyr Asp Thr Asp Ala Val Tyr Ala Leu Ile Cys Glu Leu 100 105 110 LysGln Ala Glu Phe Asp His His Ala Phe Arg Val Gly Phe Asn Ala 115 120 125Asn Leu Arg Asp Pro Asn Met Arg Tyr His Leu Ala Leu Leu Asp Gly 130 135140 Glu Val Val Gly Met Ile Gly Leu His Leu Gln Phe His Leu His His 145150 155 160 Val Asn Trp Ile Gly Glu Ile Gln Glu Leu Val Val Met Pro GlnAla 165 170 175 Arg Gly Leu Asn Val Gly Ser Lys Leu Leu Ala Trp Ala GluGlu Glu 180 185 190 Ala Arg Gln Ala Gly Ala Glu Met Thr Glu Leu Ser ThrAsn Val Lys 195 200 205 Arg His Asp Ala His Arg Phe Tyr Leu Arg Glu GlyTyr Glu Gln Ser 210 215 220 His Phe Arg Phe Thr Lys Ala Leu 225 230 13415 DNA Zea mays N_region (15)..(163) intron (164)..(322) C_region(323)..(411) 13 tctagaggat cagcatggcg cccaccgtga tgatggcctc gtcggccaccgccgtcgctc 60 cgttcctggg gctcaagtcc accgccagcc tccccgtcgc ccgccgctcctccagaagcc 120 tcggcaacgt cagcaacggc ggaaggatcc ggtgcatgca ggtaacaaatgcatcctagc 180 tagtagttct ttgcattgca gcagctgcag ctagcgagtt agtaataggaagggaactga 240 tgatccatgc atggactgat gtgtgttgcc catcccatcc catcccatttcccaaacgaa 300 ccgaaaacac cgtactacgt gcaggtgtgg ccctacggca acaagaagttcgagacgctg 360 tcgtacctgc cgccgctgtc gaccggcggg cgcatccgct gcatgcaggccatgg 415 14 174 DNA Artificial Sequence Description of ArtificialSequence chloroplast or plastid transit peptide coding sequence andamino acid sequence translation 14 atg gct tcc tct atg ctc tct tcc gctact atg gtt gcc tct ccg gct 48 Met Ala Ser Ser Met Leu Ser Ser Ala ThrMet Val Ala Ser Pro Ala 1 5 10 15 cag gcc act atg gtc gct cct ttc aacgga ctt aag tcc tcc gct gcc 96 Gln Ala Thr Met Val Ala Pro Phe Asn GlyLeu Lys Ser Ser Ala Ala 20 25 30 ttc cca gcc acc cgc aag gct aac aac gacatt act tcc atc aca agc 144 Phe Pro Ala Thr Arg Lys Ala Asn Asn Asp IleThr Ser Ile Thr Ser 35 40 45 aac ggc gga aga gtt aac tgc atg cag gcc 174Asn Gly Gly Arg Val Asn Cys Met Gln Ala 50 55 15 58 PRT ArtificialSequence 15 Met Ala Ser Ser Met Leu Ser Ser Ala Thr Met Val Ala Ser ProAla 1 5 10 15 Gln Ala Thr Met Val Ala Pro Phe Asn Gly Leu Lys Ser SerAla Ala 20 25 30 Phe Pro Ala Thr Arg Lys Ala Asn Asn Asp Ile Thr Ser IleThr Ser 35 40 45 Asn Gly Gly Arg Val Asn Cys Met Gln Ala 50 55 16 157DNA Artificial Sequence Description of Artificial Sequence syntheticoligonucleotide representing base pairs 1 through 157 of a 432 base pairAMPA acyltransferase gene 16 atggccgctt gcgagcttcg cccagccacg cagtacgacaccgacgccgt gtacgcgctg 60 atctgcgagc tcaagcaggc ggagttcgac caccacgccttccgcgtggg cttcaacgcc 120 aacctgcgcg accccaacat gcgctaccat ctggcgc 15717 187 DNA Artificial Sequence Description of Artificial Sequencesynthetic oligonucleotide sequence representing base pairs 158 through344 of a 432 base pair AMPA acyltransferase gene 17 tgcttgatggcgaagtggtc ggcatgatcg gcctgcacct ccagttccac ctgcatcatg 60 tcaactggatcggcgagatc caggagctgg tcgtgatgcc acaggcgagg ggtctgaacg 120 tcggcagcaagctcctggcg tgggccgagg aggaagccag gcaggccgga gccgagatga 180 ccgagct 18718 88 DNA Artificial Sequence Description of Artificial Sequencesynthetic oligonucleotide sequence representing base pairs 345 through432 of a 432 base pair AMPA acyltransferase gene 18 cagcaccaacgtgaagcgcc acgacgcgca ccgcttctac ctgcgcgaag gctacgagca 60 gagccacttccgcttcacca aggcgctg 88 19 432 DNA Artificial Sequence Description ofArtificial Sequence synthetic oligonucleotide providing monocotoptimized coding sequence for an AMPA acetyltransferase 19 atg gcc gcttgc gag ctt cgc cca gcc acg cag tac gac acc gac gcc 48 Met Ala Ala CysGlu Leu Arg Pro Ala Thr Gln Tyr Asp Thr Asp Ala 1 5 10 15 gtg tac gcgctg atc tgc gag ctc aag cag gcg gag ttc gac cac cac 96 Val Tyr Ala LeuIle Cys Glu Leu Lys Gln Ala Glu Phe Asp His His 20 25 30 gcc ttc cgc gtgggc ttc aac gcc aac ctg cgc gac ccc aac atg cgc 144 Ala Phe Arg Val GlyPhe Asn Ala Asn Leu Arg Asp Pro Asn Met Arg 35 40 45 tac cat ctg gcg ctgctt gat ggc gaa gtg gtc ggc atg atc ggc ctg 192 Tyr His Leu Ala Leu LeuAsp Gly Glu Val Val Gly Met Ile Gly Leu 50 55 60 cac ctc cag ttc cac ctgcat cat gtc aac tgg atc ggc gag atc cag 240 His Leu Gln Phe His Leu HisHis Val Asn Trp Ile Gly Glu Ile Gln 65 70 75 80 gag ctg gtc gtg atg ccacag gcg agg ggt ctg aac gtc ggc agc aag 288 Glu Leu Val Val Met Pro GlnAla Arg Gly Leu Asn Val Gly Ser Lys 85 90 95 ctc ctg gcg tgg gcc gag gaggaa gcc agg cag gcc gga gcc gag atg 336 Leu Leu Ala Trp Ala Glu Glu GluAla Arg Gln Ala Gly Ala Glu Met 100 105 110 acc gag ctc agc acc aac gtgaag cgc cac gac gcg cac cgc ttc tac 384 Thr Glu Leu Ser Thr Asn Val LysArg His Asp Ala His Arg Phe Tyr 115 120 125 ctg cgc gaa ggc tac gag cagagc cac ttc cgc ttc acc aag gcg ctg 432 Leu Arg Glu Gly Tyr Glu Gln SerHis Phe Arg Phe Thr Lys Ala Leu 130 135 140 20 144 PRT ArtificialSequence 20 Met Ala Ala Cys Glu Leu Arg Pro Ala Thr Gln Tyr Asp Thr AspAla 1 5 10 15 Val Tyr Ala Leu Ile Cys Glu Leu Lys Gln Ala Glu Phe AspHis His 20 25 30 Ala Phe Arg Val Gly Phe Asn Ala Asn Leu Arg Asp Pro AsnMet Arg 35 40 45 Tyr His Leu Ala Leu Leu Asp Gly Glu Val Val Gly Met IleGly Leu 50 55 60 His Leu Gln Phe His Leu His His Val Asn Trp Ile Gly GluIle Gln 65 70 75 80 Glu Leu Val Val Met Pro Gln Ala Arg Gly Leu Asn ValGly Ser Lys 85 90 95 Leu Leu Ala Trp Ala Glu Glu Glu Ala Arg Gln Ala GlyAla Glu Met 100 105 110 Thr Glu Leu Ser Thr Asn Val Lys Arg His Asp AlaHis Arg Phe Tyr 115 120 125 Leu Arg Glu Gly Tyr Glu Gln Ser His Phe ArgPhe Thr Lys Ala Leu 130 135 140 21 20 DNA Artificial SequenceDescription of Artificial Sequence synthetic oligonucleotide PHN1 foruse as an amplification primer 21 atggctgctt gtgagcttcg 20 22 20 DNAArtificial Sequence Description of Artificial Sequence syntheticoligonucleotide PHN2 for use as an amplification primer 22 cagcgccttggtgaagcgga 20 23 1630 DNA Artificial Sequence Description of ArtificialSequence expression cassette comprising plant operable promoter linkedto a coding sequence encoding an AMPA acetyltransferase linked to atranscription termination sequence 23 gcggccgcgt tcaagcttga gctcaggatttagcagcatt ccagattggg ttcaatcaac 60 aaggtacgag ccatatcact ttattcaaattggtatcgcc aaaaccaaga aggaactccc 120 atcctcaaag gtttgtaagg aagaattctcagtccaaagc ctcaacaagg tcagggtaca 180 gagtctccaa accattagcc aaaagctacaggagatcaat gaagaatctt caatcaaagt 240 aaactactgt tccagcacat gcatcatggtcagtaagttt cagaaaaaga catccaccga 300 agacttaaag ttagtgggca tctttgaaagtaatcttgtc aacatcgagc agctggcttg 360 tggggaccag acaaaaaagg aatggtgcagaattgttagg cgcacctacc aaaagcatct 420 ttgcctttat tgcaaagata aagcagattcctctagtaca agtggggaac aaaataacgt 480 ggaaaagagc tgtcctgaca gcccactcactaatgcgtat gacgaacgca gtgacgacca 540 caaaagaatt ccctctatat aagaaggcattcattcccat ttgaaggatc atcagatact 600 gaaccaatcc ttctagaaga tctccacaatggcttcctct atgctctctt ccgctactat 660 ggttgcctct ccggctcagg ccactatggtcgctcctttc aacggactta agtcctccgc 720 tgccttccca gccacccgca aggctaacaacgacattact tccatcacaa gcaacggcgg 780 aagagttaac tgcatgcagg tgtggcctccgattggaaag aagaagtttg agactctctc 840 ttaccttcct gaccttaccg attccggtggtcgcgtcaac tgcatgcagg cc atg gct 898 Met Ala 1 gct tgt gag ctt cgc ccggcc acg cag tac gac acc gac gcg gtt tac 946 Ala Cys Glu Leu Arg Pro AlaThr Gln Tyr Asp Thr Asp Ala Val Tyr 5 10 15 gcg ctg att tgt gag cta aaacag gcg gag ttt gac cac cac gcg ttt 994 Ala Leu Ile Cys Glu Leu Lys GlnAla Glu Phe Asp His His Ala Phe 20 25 30 cgc gtg ggt ttt aac gcc aat ctgcgc gac cca aac atg cgc tac cat 1042 Arg Val Gly Phe Asn Ala Asn Leu ArgAsp Pro Asn Met Arg Tyr His 35 40 45 50 ctg gcg ctg ctt gat ggc gaa gttgtc ggc atg atc ggc ctg cat ttg 1090 Leu Ala Leu Leu Asp Gly Glu Val ValGly Met Ile Gly Leu His Leu 55 60 65 cag ttt cat ctg cat cat gtc aac tggatc ggc gaa att cag gag ttg 1138 Gln Phe His Leu His His Val Asn Trp IleGly Glu Ile Gln Glu Leu 70 75 80 gtg gta atg ccg cag gcg cgc ggt ctg aacgtc ggc agt aag tta ctg 1186 Val Val Met Pro Gln Ala Arg Gly Leu Asn ValGly Ser Lys Leu Leu 85 90 95 gcg tgg gca gaa gaa gaa gcc cgc cag gcc ggggcc gaa atg acc gaa 1234 Ala Trp Ala Glu Glu Glu Ala Arg Gln Ala Gly AlaGlu Met Thr Glu 100 105 110 ctt tcg acc aac gtg aag cgc cac gac gcg caccgt ttc tat ctg cgc 1282 Leu Ser Thr Asn Val Lys Arg His Asp Ala His ArgPhe Tyr Leu Arg 115 120 125 130 gaa ggc tac gag cag agc cac ttc cgc ttcacc aag gcg ctg 1324 Glu Gly Tyr Glu Gln Ser His Phe Arg Phe Thr Lys AlaLeu 135 140 taatgagctc ggtaccggat ccaattcccg atcgttcaaa catttggcaataaagtttct 1384 taagattgaa tcctgttgcc ggtcttgcga tgattatcat ataatttctgttgaattacg 1444 ttaagcatgt aataattaac atgtaatgca tgacgttatt tatgagatgggtttttatga 1504 ttagagtccc gcaattatac atttaatacg cgatagaaaa caaaatatagcgcgcaaact 1564 aggataaatt atcgcgcgcg gtgtcatcta tgttactaga tcggggatcgatccccgggc 1624 ggccgc 1630 24 144 PRT Artificial Sequence 24 Met AlaAla Cys Glu Leu Arg Pro Ala Thr Gln Tyr Asp Thr Asp Ala 1 5 10 15 ValTyr Ala Leu Ile Cys Glu Leu Lys Gln Ala Glu Phe Asp His His 20 25 30 AlaPhe Arg Val Gly Phe Asn Ala Asn Leu Arg Asp Pro Asn Met Arg 35 40 45 TyrHis Leu Ala Leu Leu Asp Gly Glu Val Val Gly Met Ile Gly Leu 50 55 60 HisLeu Gln Phe His Leu His His Val Asn Trp Ile Gly Glu Ile Gln 65 70 75 80Glu Leu Val Val Met Pro Gln Ala Arg Gly Leu Asn Val Gly Ser Lys 85 90 95Leu Leu Ala Trp Ala Glu Glu Glu Ala Arg Gln Ala Gly Ala Glu Met 100 105110 Thr Glu Leu Ser Thr Asn Val Lys Arg His Asp Ala His Arg Phe Tyr 115120 125 Leu Arg Glu Gly Tyr Glu Gln Ser His Phe Arg Phe Thr Lys Ala Leu130 135 140 25 2122 DNA Artificial Sequence Description of ArtificialSequenceexpression cassette comprising plant promoter linked to sequenceencoding AMPA acetyl transferase linked to termination sequence 25ctgcaggtcc gatgtgagac ttttcaacaa agggtaatat ccggaaacct cctcggattc 60cattgcccag ctatctgtca ctttattgtg aagatagtgg aaaaggaagg tggctcctac 120aaatgccatc attgcgataa aggaaaggcc atcgttgaag atgcctctgc cgacagtggt 180cccaaagatg gacccccacc cacgaggagc atcgtggaaa aagaagacgt tccaaccacg 240tcttcaaagc aagtggattg atgtgatggt ccgatgtgag acttttcaac aaagggtaat 300atccggaaac ctcctcggat tccattgccc agctatctgt cactttattg tgaagatagt 360ggaaaaggaa ggtggctcct acaaatgcca tcattgcgat aaaggaaagg ccatcgttga 420agatgcctct gccgacagtg gtcccaaaga tggaccccca cccacgagga gcatcgtgga 480aaaagaagac gttccaacca cgtcttcaaa gcaagtggat tgatgtgata tctccactga 540cgtaagggat gacgcacaat cccactatcc ttcgcaagac ccttcctcta tataaggaag 600ttcatttcat ttggagagga cacgctgaca agctgactct agcagatcct ctagaaccat 660cttccacaca ctcaagccac actattggag aacacacagg gacaacacac cataagatcc 720aagggaggcc tccgccgccg ccggtaacca ccccgcccct ctcctctttc tttctccgtt 780tttttttccg tctcggtctc gatctttggc cttggtagtt tgggtgggcg agaggcggct 840tcgtgcgcgc ccagatcggt gcgcgggagg ggcgggatct cgcggggaat ggggctctcg 900gatgtagatc tgcgatccgc cgttgttggg ggagatgatg gggcgtttaa aatttcgccg 960tgctaaacaa gatcaggaag aggggaaaag ggcactatgg tttatatttt tatatatttc 1020tgctgcttcg tcaggcttag atgtgctaga tctttctttc ttctttttgt gggtagaatt 1080taatccctca gcattgttca tcggtagttt ttcttttcat gatttcgtga caaatgcagc 1140ctcgtgcgga gcttttttgt aggtagaagt gatcaaccat ggcgcaagtt agcagaatct 1200gcaatggtgt gcagaaccca tctcttatct ccaatctctc gaaatccagt caacgcaaat 1260ctcccttatc ggtttctctg aagacgcagc agcatccacg agcttatccg atttcgtcgt 1320cgtggggatt gaagaagagt gggatgacgt taattggctc tgagcttcgt cctcttaagg 1380tcatgtcttc tgtttccacg gcgtgc atg gcc gct tgc gag ctt cgc cca gcc 1433Met Ala Ala Cys Glu Leu Arg Pro Ala 1 5 acg cag tac gac acc gac gcc gtgtac gcg ctg atc tgc gag ctc aag 1481 Thr Gln Tyr Asp Thr Asp Ala Val TyrAla Leu Ile Cys Glu Leu Lys 10 15 20 25 cag gcg gag ttc gac cac cac gccttc cgc gtg ggc ttc aac gcc aac 1529 Gln Ala Glu Phe Asp His His Ala PheArg Val Gly Phe Asn Ala Asn 30 35 40 ctg cgc gac ccc aac atg cgc tac catctg gcg ctg ctt gat ggc gaa 1577 Leu Arg Asp Pro Asn Met Arg Tyr His LeuAla Leu Leu Asp Gly Glu 45 50 55 gtg gtc ggc atg atc ggc ctg cac ctc cagttc cac ctg cat cat gtc 1625 Val Val Gly Met Ile Gly Leu His Leu Gln PheHis Leu His His Val 60 65 70 aac tgg atc ggc gag atc cag gag ctg gtc gtgatg cca cag gcg agg 1673 Asn Trp Ile Gly Glu Ile Gln Glu Leu Val Val MetPro Gln Ala Arg 75 80 85 ggt ctg aac gtc ggc agc aag ctc ctg gcg tgg gccgag gag gaa gcc 1721 Gly Leu Asn Val Gly Ser Lys Leu Leu Ala Trp Ala GluGlu Glu Ala 90 95 100 105 agg cag gcc gga gcc gag atg acc gag ctc agcacc aac gtg aag cgc 1769 Arg Gln Ala Gly Ala Glu Met Thr Glu Leu Ser ThrAsn Val Lys Arg 110 115 120 cac gac gcg cac cgc ttc tac ctg cgc gaa ggctac gag cag agc cac 1817 His Asp Ala His Arg Phe Tyr Leu Arg Glu Gly TyrGlu Gln Ser His 125 130 135 ttc cgc ttc acc aag gcg ctg taaagatctgaattctgcat gcgtttggac 1868 Phe Arg Phe Thr Lys Ala Leu 140 gtatgctcattcaggttgga gccaatttgg ttgatgtgtg tgcgagttct tgcgagtctg 1928 atgagacatctctgtattgt gtttctttcc ccagtgtttt ctgtacttgt gtaatcggct 1988 aatcgccaacagattcggcg atgaataaat gagaaataaa ttgttctgat tttgagtgca 2048 aaaaaaaaggaattagatct gtgtgtgttt tttggatccc cggggcggcc gccccgggtg 2108 gtgagcttctgcag 2122 26 144 PRT Artificial Sequence 26 Met Ala Ala Cys Glu Leu ArgPro Ala Thr Gln Tyr Asp Thr Asp Ala 1 5 10 15 Val Tyr Ala Leu Ile CysGlu Leu Lys Gln Ala Glu Phe Asp His His 20 25 30 Ala Phe Arg Val Gly PheAsn Ala Asn Leu Arg Asp Pro Asn Met Arg 35 40 45 Tyr His Leu Ala Leu LeuAsp Gly Glu Val Val Gly Met Ile Gly Leu 50 55 60 His Leu Gln Phe His LeuHis His Val Asn Trp Ile Gly Glu Ile Gln 65 70 75 80 Glu Leu Val Val MetPro Gln Ala Arg Gly Leu Asn Val Gly Ser Lys 85 90 95 Leu Leu Ala Trp AlaGlu Glu Glu Ala Arg Gln Ala Gly Ala Glu Met 100 105 110 Thr Glu Leu SerThr Asn Val Lys Arg His Asp Ala His Arg Phe Tyr 115 120 125 Leu Arg GluGly Tyr Glu Gln Ser His Phe Arg Phe Thr Lys Ala Leu 130 135 140 27 2378DNA Artificial Sequence Description of Artificial Sequenceexpressioncassette comprising a plant promoter linked to an intron, a sequenceencoding an AMPA acetyl transferase, and a termination sequence 27gatatcccta gggcggccgc gttaacaagc ttactcgagg tcattcatat gcttgagaag 60agagtcggga tagtccaaaa taaaacaaag gtaagattac ctggtcaaaa gtgaaaacat 120cagttaaaag gtggtataaa gtaaaatatc ggtaataaaa ggtggcccaa agtgaaattt 180actcttttct actattataa aaattgagga tgtttttgtc ggtactttga tacgtcattt 240ttgtatgaat tggtttttaa gtttattcgc ttttggaaat gcatatctgt atttgagtcg 300ggttttaagt tcgtttgctt ttgtaaatac agagggattt gtataagaaa tatctttaga 360aaaacccata tgctaatttg acataatttt tgagaaaaat atatattcag gcgaattctc 420acaatgaaca ataataagat taaaatagct ttcccccgtt gcagcgcatg ggtatttttt 480ctagtaaaaa taaaagataa acttagactc aaaacattta caaaaacaac ccctaaagtt 540cctaaagccc aaagtgctat ccacgatcca tagcaagccc agcccaaccc aacccaaccc 600agcccacccc agtccagcca actggacaat agtctccaca cccccccact atcaccgtga 660gttgtccgca cgcaccgcac gtctcgcagc caaaaaaaaa aagaaagaaa aaaaagaaaa 720agaaaaaaca gcaggtgggt ccgggtcgtg ggggccggaa acgcgaggag gatcgcgagc 780cagcgacgag gccggccctc cctccgcttc caaagaaacg ccccccatcg ccactatata 840catacccccc cctctcctcc catcccccca accctaccac caccaccacc accacctcca 900cctcctcccc cctcgctgcc ggacgacgag ctcctccccc ctccccctcc gccgccgccg 960cgccggtaac caccccgccc ctctcctctt tctttctccg tttttttttc cgtctcggtc 1020tcgatctttg gccttggtag tttgggtggg cgagaggcgg cttcgtgccg cccagatcgg 1080tgcgcgggag gggcgggatc tcgcggctgg ctctcgcccc cgtggatccg gcccggatct 1140cgcggggaat ggggctctcg gatgtagatc tgcgatccgc cgttgttggg gccgatgatg 1200gggcccttaa aatttccgcc gtgctaaaca agatcaggaa gaggggaaaa gggcactatg 1260gtttatattt ttatatattt ctgctgcttc gtcaggctta gatgtgctag atctttcttt 1320cttctttttg tgggtagaat ttaatccctc agcattgttc atcggtagtt tttcttttca 1380tgattcgtga caaatgcagc ctcgtgcgga cgtttttttg taggtagaag tgatcaacca 1440tggcgcaagt tagcagaatc tgcaatggtg tgcagaaccc atctcttatc tccaatctct 1500cgaaatccag tcaacgcaaa tctcccttat cggtttctct gaagacgcag cagcatccac 1560gagcttatcc gatttcgtcg tcgtggggat tgaagaagag tgggatgacg ttaattggct 1620ctgagcttcg tcctcttaag gtcatgtctt ctgtttccac ggcgtgc atg gcc gct 1676 MetAla Ala 1 tgc gag ctt cgc cca gcc acg cag tac gac acc gac gcc gtg tacgcg 1724 Cys Glu Leu Arg Pro Ala Thr Gln Tyr Asp Thr Asp Ala Val Tyr Ala5 10 15 ctg atc tgc gag ctc aag cag gcg gag ttc gac cac cac gcc ttc cgc1772 Leu Ile Cys Glu Leu Lys Gln Ala Glu Phe Asp His His Ala Phe Arg 2025 30 35 gtg ggc ttc aac gcc aac ctg cgc gac ccc aac atg cgc tac cat ctg1820 Val Gly Phe Asn Ala Asn Leu Arg Asp Pro Asn Met Arg Tyr His Leu 4045 50 gcg ctg ctt gat ggc gaa gtg gtc ggc atg atc ggc ctg cac ctc cag1868 Ala Leu Leu Asp Gly Glu Val Val Gly Met Ile Gly Leu His Leu Gln 5560 65 ttc cac ctg cat cat gtc aac tgg atc ggc gag atc cag gag ctg gtc1916 Phe His Leu His His Val Asn Trp Ile Gly Glu Ile Gln Glu Leu Val 7075 80 gtg atg cca cag gcg agg ggt ctg aac gtc ggc agc aag ctc ctg gcg1964 Val Met Pro Gln Ala Arg Gly Leu Asn Val Gly Ser Lys Leu Leu Ala 8590 95 tgg gcc gag gag gaa gcc agg cag gcc gga gcc gag atg acc gag ctc2012 Trp Ala Glu Glu Glu Ala Arg Gln Ala Gly Ala Glu Met Thr Glu Leu 100105 110 115 agc acc aac gtg aag cgc cac gac gcg cac cgc ttc tac ctg cgcgaa 2060 Ser Thr Asn Val Lys Arg His Asp Ala His Arg Phe Tyr Leu Arg Glu120 125 130 ggc tac gag cag agc cac ttc cgc ttc acc aag gcg ctgtaaagatctg 2109 Gly Tyr Glu Gln Ser His Phe Arg Phe Thr Lys Ala Leu 135140 aattcccgat cgttcaaaca tttggcaata aagtttctta agattgaatc ctgttgccgg2169 tcttgcgatg attatcatat aatttctgtt gaattacgtt aagcatgtaa taattaacat2229 gtaatgcatg acgttattta tgagatgggt ttttatgatt agagtcccgc aattatacat2289 ttaatacgcg atagaaaaca aaatatagcg cgcaaactag gataaattat cgcgcgcggt2349 gtcatctatg ttactagatc ggggatatc 2378 28 144 PRT Artificial Sequence28 Met Ala Ala Cys Glu Leu Arg Pro Ala Thr Gln Tyr Asp Thr Asp Ala 1 510 15 Val Tyr Ala Leu Ile Cys Glu Leu Lys Gln Ala Glu Phe Asp His His 2025 30 Ala Phe Arg Val Gly Phe Asn Ala Asn Leu Arg Asp Pro Asn Met Arg 3540 45 Tyr His Leu Ala Leu Leu Asp Gly Glu Val Val Gly Met Ile Gly Leu 5055 60 His Leu Gln Phe His Leu His His Val Asn Trp Ile Gly Glu Ile Gln 6570 75 80 Glu Leu Val Val Met Pro Gln Ala Arg Gly Leu Asn Val Gly Ser Lys85 90 95 Leu Leu Ala Trp Ala Glu Glu Glu Ala Arg Gln Ala Gly Ala Glu Met100 105 110 Thr Glu Leu Ser Thr Asn Val Lys Arg His Asp Ala His Arg PheTyr 115 120 125 Leu Arg Glu Gly Tyr Glu Gln Ser His Phe Arg Phe Thr LysAla Leu 130 135 140 29 2107 DNA Artificial Sequence Description ofArtificial Sequenceexpression cassette comprising plant operablepromoter linked to a leader, intron, a sequence encoding an AMPAacetyltransferase, and termination sequence 29 gcggccgcgt taacaagcttctgcaggtcc gatgtgagac ttttcaacaa agggtaatat 60 ccggaaacct cctcggattccattgcccag ctatctgtca ctttattgtg aagatagtgg 120 aaaaggaagg tggctcctacaaatgccatc attgcgataa aggaaaggcc atcgttgaag 180 atgcctctgc cgacagtggtcccaaagatg gacccccacc cacgaggagc atcgtggaaa 240 aagaagacgt tccaaccacgtcttcaaagc aagtggattg atgtgatggt ccgatgtgag 300 acttttcaac aaagggtaatatccggaaac ctcctcggat tccattgccc agctatctgt 360 cactttattg tgaagatagtggaaaaggaa ggtggctcct acaaatgcca tcattgcgat 420 aaaggaaagg ccatcgttgaagatgcctct gccgacagtg gtcccaaaga tggaccccca 480 cccacgagga gcatcgtggaaaaagaagac gttccaacca cgtcttcaaa gcaagtggat 540 tgatgtgata tctccactgacgtaagggat gacgcacaat cccactatcc ttcgcaagac 600 ccttcctcta tataaggaagttcatttcat ttggagagga cacgctgaca agctgactct 660 agcagatcct ctagaaccatcttccacaca ctcaagccac actattggag aacacacagg 720 gacaacacac cataagatccaagggaggcc tccgccgccg ccggtaacca ccccgcccct 780 ctcctctttc tttctccgtttttttttccg tctcggtctc gatctttggc cttggtagtt 840 tgggtgggcg agaggcggcttcgtgcgcgc ccagatcggt gcgcgggagg ggcgggatct 900 cgcggggaat ggggctctcggatgtagatc tgcgatccgc cgttgttggg ggagatgatg 960 gggcgtttaa aatttcgccgtgctaaacaa gatcaggaag aggggaaaag ggcactatgg 1020 tttatatttt tatatatttctgctgcttcg tcaggcttag atgtgctaga tctttctttc 1080 ttctttttgt gggtagaatttaatccctca gcattgttca tcggtagttt ttcttttcat 1140 gatttcgtga caaatgcagcctcgtgcgga gcttttttgt aggtagaagt gatcaaccat 1200 ggcgcaagtt agcagaatctgcaatggtgt gcagaaccca tctcttatct ccaatctctc 1260 gaaatccagt caacgcaaatctcccttatc ggtttctctg aagacgcagc agcatccacg 1320 agcttatccg atttcgtcgtcgtggggatt gaagaagagt gggatgacgt taattggctc 1380 tgagcttcgt cctcttaaggtcatgtcttc tgtttccacg gcgtgc atg gcc gct 1435 Met Ala Ala 1 tgc gag cttcgc cca gcc acg cag tac gac acc gac gcc gtg tac gcg 1483 Cys Glu Leu ArgPro Ala Thr Gln Tyr Asp Thr Asp Ala Val Tyr Ala 5 10 15 ctg atc tgc gagctc aag cag gcg gag ttc gac cac cac gcc ttc cgc 1531 Leu Ile Cys Glu LeuLys Gln Ala Glu Phe Asp His His Ala Phe Arg 20 25 30 35 gtg ggc ttc aacgcc aac ctg cgc gac ccc aac atg cgc tac cat ctg 1579 Val Gly Phe Asn AlaAsn Leu Arg Asp Pro Asn Met Arg Tyr His Leu 40 45 50 gcg ctg ctt gat ggcgaa gtg gtc ggc atg atc ggc ctg cac ctc cag 1627 Ala Leu Leu Asp Gly GluVal Val Gly Met Ile Gly Leu His Leu Gln 55 60 65 ttc cac ctg cat cat gtcaac tgg atc ggc gag atc cag gag ctg gtc 1675 Phe His Leu His His Val AsnTrp Ile Gly Glu Ile Gln Glu Leu Val 70 75 80 gtg atg cca cag gcg agg ggtctg aac gtc ggc agc aag ctc ctg gcg 1723 Val Met Pro Gln Ala Arg Gly LeuAsn Val Gly Ser Lys Leu Leu Ala 85 90 95 tgg gcc gag gag gaa gcc agg caggcc gga gcc gag atg acc gag ctc 1771 Trp Ala Glu Glu Glu Ala Arg Gln AlaGly Ala Glu Met Thr Glu Leu 100 105 110 115 agc acc aac gtg aag cgc cacgac gcg cac cgc ttc tac ctg cgc gaa 1819 Ser Thr Asn Val Lys Arg His AspAla His Arg Phe Tyr Leu Arg Glu 120 125 130 ggc tac gag cag agc cac ttccgc ttc acc aag gcg ctg taaagatctg 1868 Gly Tyr Glu Gln Ser His Phe ArgPhe Thr Lys Ala Leu 135 140 aattctgcat gcgtttggac gtatgctcat tcaggttggagccaatttgg ttgatgtgtg 1928 tgcgagttct tgcgagtctg atgagacatc tctgtattgtgtttctttcc ccagtgtttt 1988 ctgtacttgt gtaatcggct aatcgccaac agattcggcgatgaataaat gagaaataaa 2048 ttgttctgat tttgagtgca aaaaaaaagg aattagatctgtgtgtgttt tttggatcc 2107 30 144 PRT Artificial Sequence 30 Met Ala AlaCys Glu Leu Arg Pro Ala Thr Gln Tyr Asp Thr Asp Ala 1 5 10 15 Val TyrAla Leu Ile Cys Glu Leu Lys Gln Ala Glu Phe Asp His His 20 25 30 Ala PheArg Val Gly Phe Asn Ala Asn Leu Arg Asp Pro Asn Met Arg 35 40 45 Tyr HisLeu Ala Leu Leu Asp Gly Glu Val Val Gly Met Ile Gly Leu 50 55 60 His LeuGln Phe His Leu His His Val Asn Trp Ile Gly Glu Ile Gln 65 70 75 80 GluLeu Val Val Met Pro Gln Ala Arg Gly Leu Asn Val Gly Ser Lys 85 90 95 LeuLeu Ala Trp Ala Glu Glu Glu Ala Arg Gln Ala Gly Ala Glu Met 100 105 110Thr Glu Leu Ser Thr Asn Val Lys Arg His Asp Ala His Arg Phe Tyr 115 120125 Leu Arg Glu Gly Tyr Glu Gln Ser His Phe Arg Phe Thr Lys Ala Leu 130135 140 31 2436 DNA Artificial Sequence Description of ArtificialSequencemonocot expression cassette comprising plant operable promoterlinked to an intron, a sequence coding for an AMPA acetyltransferase,and a termination sequence 31 gcggccgcgt taacaagctt ctgcaggtccgatgtgagac ttttcaacaa agggtaatat 60 ccggaaacct cctcggattc cattgcccagctatctgtca ctttattgtg aagatagtgg 120 aaaaggaagg tggctcctac aaatgccatcattgcgataa aggaaaggcc atcgttgaag 180 atgcctctgc cgacagtggt cccaaagatggacccccacc cacgaggagc atcgtggaaa 240 aagaagacgt tccaaccacg tcttcaaagcaagtggattg atgtgatggt ccgatgtgag 300 acttttcaac aaagggtaat atccggaaacctcctcggat tccattgccc agctatctgt 360 cactttattg tgaagatagt ggaaaaggaaggtggctcct acaaatgcca tcattgcgat 420 aaaggaaagg ccatcgttga agatgcctctgccgacagtg gtcccaaaga tggaccccca 480 cccacgagga gcatcgtgga aaaagaagacgttccaacca cgtcttcaaa gcaagtggat 540 tgatgtgata tctccactga cgtaagggatgacgcacaat cccactatcc ttcgcaagac 600 ccttcctcta tataaggaag ttcatttcatttggagagga cacgctgaca agctgactct 660 agcagatcta ccgtcttcgg tacgcgctcactccgccctc tgcctttgtt actgccacgt 720 ttctctgaat gctctcttgt gtggtgattgctgagagtgg tttagctgga tctagaatta 780 cactctgaaa tcgtgttctg cctgtgctgattacttgccg tcctttgtag cagcaaaata 840 tagggacatg gtagtacgaa acgaagatagaacctacaca gcaatacgag aaatgtgtaa 900 tttggtgctt agcggtattt atttaagcacatgttggtgt tatagggcac ttggattcag 960 aagtttgctg ttaatttagg cacaggcttcatactacatg ggtcaatagt atagggattc 1020 atattatagg cgatactata ataatttgttcgtctgcaga gcttattatt tgccaaaatt 1080 agatattcct attctgtttt tgtttgtgtgctgttaaatt gttaacgcct gaaggaataa 1140 atataaatga cgaaattttg atgtttatctctgctccttt attgtgacca taagtcaaga 1200 tcagatgcac ttgttttaaa tattgttgtctgaagaaata agtactgaca gtattttgat 1260 gcattgatct gcttgtttgt tgtaacaaaatttaaaaata aagagtttcc tttttgttgc 1320 tctccttacc tcctgatggt atctagtatctaccaactga cactatattg cttctcttta 1380 catacgtatc ttgctcgatg ccttctccctagtgttgacc agtgttactc acatagtctt 1440 tgctcatttc attgtaatgc agataccaagcggcctctag aggatccagg agcaaccatg 1500 gcgcaagtta gcagaatctg caatggtgtgcagaacccat ctcttatctc caatctctcg 1560 aaatccagtc aacgcaaatc tcccttatcggtttctctga agacgcagca gcatccacga 1620 gcttatccga tttcgtcgtc gtggggattgaagaagagtg ggatgacgtt aattggctct 1680 gagcttcgtc ctcttaaggt catgtcttctgtttccacgg cgtgc atg gcc gct tgc 1737 Met Ala Ala Cys 1 gag ctt cgc ccagcc acg cag tac gac acc gac gcc gtg tac gcg ctg 1785 Glu Leu Arg Pro AlaThr Gln Tyr Asp Thr Asp Ala Val Tyr Ala Leu 5 10 15 20 atc tgc gag ctcaag cag gcg gag ttc gac cac cac gcc ttc cgc gtg 1833 Ile Cys Glu Leu LysGln Ala Glu Phe Asp His His Ala Phe Arg Val 25 30 35 ggc ttc aac gcc aacctg cgc gac ccc aac atg cgc tac cat ctg gcg 1881 Gly Phe Asn Ala Asn LeuArg Asp Pro Asn Met Arg Tyr His Leu Ala 40 45 50 ctg ctt gat ggc gaa gtggtc ggc atg atc ggc ctg cac ctc cag ttc 1929 Leu Leu Asp Gly Glu Val ValGly Met Ile Gly Leu His Leu Gln Phe 55 60 65 cac ctg cat cat gtc aac tggatc ggc gag atc cag gag ctg gtc gtg 1977 His Leu His His Val Asn Trp IleGly Glu Ile Gln Glu Leu Val Val 70 75 80 atg cca cag gcg agg ggt ctg aacgtc ggc agc aag ctc ctg gcg tgg 2025 Met Pro Gln Ala Arg Gly Leu Asn ValGly Ser Lys Leu Leu Ala Trp 85 90 95 100 gcc gag gag gaa gcc agg cag gccgga gcc gag atg acc gag ctc agc 2073 Ala Glu Glu Glu Ala Arg Gln Ala GlyAla Glu Met Thr Glu Leu Ser 105 110 115 acc aac gtg aag cgc cac gac gcgcac cgc ttc tac ctg cgc gaa ggc 2121 Thr Asn Val Lys Arg His Asp Ala HisArg Phe Tyr Leu Arg Glu Gly 120 125 130 tac gag cag agc cac ttc cgc ttcacc aag gcg ctg taaagatctg 2167 Tyr Glu Gln Ser His Phe Arg Phe Thr LysAla Leu 135 140 aattcccgat cgttcaaaca tttggcaata aagtttctta agattgaatcctgttgccgg 2227 tcttgcgatg attatcatat aatttctgtt gaattacgtt aagcatgtaataattaacat 2287 gtaatgcatg acgttattta tgagatgggt ttttatgatt agagtcccgcaattatacat 2347 ttaatacgcg atagaaaaca aaatatagcg cgcaaactag gataaattatcgcgcgcggt 2407 gtcatctatg ttactagatc ggggatatc 2436 32 144 PRTArtificial Sequence 32 Met Ala Ala Cys Glu Leu Arg Pro Ala Thr Gln TyrAsp Thr Asp Ala 1 5 10 15 Val Tyr Ala Leu Ile Cys Glu Leu Lys Gln AlaGlu Phe Asp His His 20 25 30 Ala Phe Arg Val Gly Phe Asn Ala Asn Leu ArgAsp Pro Asn Met Arg 35 40 45 Tyr His Leu Ala Leu Leu Asp Gly Glu Val ValGly Met Ile Gly Leu 50 55 60 His Leu Gln Phe His Leu His His Val Asn TrpIle Gly Glu Ile Gln 65 70 75 80 Glu Leu Val Val Met Pro Gln Ala Arg GlyLeu Asn Val Gly Ser Lys 85 90 95 Leu Leu Ala Trp Ala Glu Glu Glu Ala ArgGln Ala Gly Ala Glu Met 100 105 110 Thr Glu Leu Ser Thr Asn Val Lys ArgHis Asp Ala His Arg Phe Tyr 115 120 125 Leu Arg Glu Gly Tyr Glu Gln SerHis Phe Arg Phe Thr Lys Ala Leu 130 135 140

What is claimed is:
 1. A recombinant plant transformed with apolynucleotide sequence comprising: a) a plant functional promotersequence operably linked to; b) a structural DNA sequence which encodesan acyltransferase enzyme, operably linked to; c) a 3′ sequence whichfunctions in plants to cause transcription termination; wherein thepromoter sequence causes sufficient expression of said enzyme in planttissue to enhance the phosphonate herbicide tolerance of a planttransformed with said polynucleotide sequence, and wherein said enzymetransfers an acyl group from an acylated donor compound to the terminalamine of a phosphonate herbicide.
 2. The plant of claim 1 in which saidacyltransferase enzyme is localized to plastids in said plant.
 3. Theplant of claim 2 wherein said plastids comprise chloroplasts.
 4. Theplant of claim 3 in which said structural DNA sequence comprises a 5′sequence encoding an amino-terminal chloroplast transit peptide operablylinked 5′ to said structural DNA sequence, wherein said enzyme islocalized to the plant chloroplasts or plastids.
 5. The plant of claim2, wherein said acylated donor is an acyl coenzyme A.
 6. The plant ofclaim 5 wherein said acyl coenzyme A is selected from the groupconsisting of acetyl coenzyme A, propionyl coenzyme A, malonyl coenzymeA, succinyl coenzyme A, and methyl-malonyl coenzyme A.
 7. The plant ofclaim 6 wherein said acyl coenzyme A is acetyl coenzyme A.
 8. The plantof claim 6 selected from the group consisting of corn, wheat, cotton,rice, soybean, sugarbeet, canola, flax, barley, oilseed rape, sunflower,potato, tobacco, tomato, alfalfa, lettuce, apple, poplar, pine,eucalyptus, acacia, poplar, sweetgum, radiata pine, loblolly pine,spruce, teak, alfalfa, clovers and other forage crops, turf grasses,oilpalm, sugarcane, banana, coffee, tea, cacao, apples, walnuts,almonds, grapes, peanuts, pulses, petunia, marigolds, vinca, begonias,geraniums, pansy, impatiens, oats, sorghum, and millet.
 9. The plant ofclaim 2 in which the promoter sequence is derived from a plant DNA viruspromoter sequence.
 10. The plant of claim 9 in which said promotersequence is selected from the group consisting of CaMV35S, FMV35S,enhanced CaMV35S, enhanced FMV35S, comalina yellow mottle viruspromoter, and sugar cane bacilliform DNA virus promoter.
 11. The plantof claim 10 in which the structural DNA sequence is or is complementaryto an E. coli phnO gene sequence as set forth in SEQ ID NO:
 3. 12. Theplant of claim 11 wherein said structural DNA sequence encodes a peptideselected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:
 8. 13.The plant of claim 12 in which the structural DNA sequence is derivedfrom a microbe, wherein said sequence is or is complementary to apolynucleotide sequence capable of hybridizing to a sequence selectedfrom the group consisting of SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11,SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO:
 19. 14. Theplant of claim 13 in which the acyltransferase enzyme is substantiallysimilar to an E. coli PhnO peptide which functions in plants to transferan acyl group from an acylated donor compound to the terminal amine of aphosphonate herbicide.
 15. The plant of claim 14 wherein saidphosphonate herbicide is selected from the group consisting ofglyphosate and AMPA.
 16. The plant of claim 4 in which the 5′ sequenceencoding an amino-terminal chloroplast transit peptide is selected fromthe group consisting of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, andSEQ ID NO:
 14. 17. The plant according to claim 3 which exhibitsenhanced tolerance to one or more phosphonate herbicides, the herbicidebeing selected from the group consisting of glyphosate and AMPA.
 18. Aseed produced from the plant of claim 17, wherein said seed comprisessaid polynucleotide sequence.
 19. A plant grown from the seed of claim18.
 20. A stably transformed phosphonate herbicide tolerant recombinantplant which contains a polynucleotide sequence comprising: a) a plantfunctional promoter sequence operably linked to; b) a structural DNAsequence which encodes an acyltransferase enzyme, operably linked to; c)a 3′ sequence which functions in plants to cause transcriptiontermination; wherein the promoter sequence is heterologous with respectto the structural DNA sequence and causes sufficient expression of saidenzyme in plant tissue to enhance the phosphonate herbicide tolerance ofa plant transformed with said polynucleotide sequence, wherein saidenzyme transfers an acyl group from an acylated donor to the terminalamine of a phosphonate herbicide, and wherein said plant expresses a GOXgene which encodes a plant functional glyphosate oxidoreductase enzyme.21. A method for selectively enhancing phosphonate herbicide tolerancein a recombinant plant comprising the steps of: a) transforming saidplant with a polynucleotide sequence comprising i) a promoter sequencewhich functions in plants to cause the production of an RNA sequence,operably linked to; ii) a structural DNA sequence capable of producingan RNA sequence which encodes an acyltransferase enzyme, operably linkedto; iii) a 3′ non-translated sequence which functions in plants to causethe addition of a polyadenylated nucleotide sequence to the 3′ end ofsaid RNA sequence; wherein the promoter sequence is heterologous withrespect to the structural DNA sequence and causes sufficient expressionof said enzyme in plant tissue to enhance the phosphonate herbicidetolerance of a plant transformed with said polynucleotide sequence,wherein said enzyme transfers an acyl group from an acylated donorsubstrate to the terminal amine of a phosphonate herbicide substrate,and wherein said plant expresses a GOX gene which encodes a plantfunctional glyphosate oxidoreductase enzyme; and b) expressing aherbicide tolerant effective amount of said acyltransferase enzyme insaid plant.
 22. The method according to claim 21, wherein saidacyltransferase is expressed from a DNA sequence selected from the groupconsisting of SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11, and SEQ ID NO:19.
 23. A method for producing a genetically transformed phosphonateherbicide tolerant plant comprising the steps of: a) inserting into thegenome of a plant cell a polynucleotide sequence comprising; i) apromoter sequence which functions in plant cells to cause the productionof an RNA sequence, operably linked to; ii) a structural DNA sequencecapable of producing an RNA sequence which encodes an acyltransferaseenzyme which transfers an acyl group from an acylated donor to theterminal amine of a phosphonate herbicide substrate, operably linked to;iii) a 3′ non-translated sequence which functions in plant cells tocause the addition of a polyadenylated nucleotide sequence to the 3′ endof said RNA sequence; wherein the promoter sequence is heterologous withrespect to the structural DNA sequence and causes sufficient expressionof said enzyme in a plant cell to enhance the phosphonate herbicidetolerance of a plant cell transformed with said polynucleotide sequence;b) selecting a transformed plant cell; and c) regenerating from thetransformed plant cell a genetically transformed plant which exhibitsimproved phosphonate herbicide tolerance.
 24. A method for producing agenetically transformed phosphonate herbicide tolerant plant comprisingthe steps of: a) inserting into the genome of a plant cell apolynucleotide sequence comprising: i) a promoter sequence whichfunctions in plant cells to cause the production of an RNA sequence,operably linked to; ii) a structural DNA sequence capable of producingan RNA sequence which encodes an acyltransferase enzyme which transfersan acyl group from an acylated donor to the terminal amine of aphosphonate herbicide substrate, operably linked to; iii) a 3′non-translated sequence which functions in plant cells to cause theaddition of a polyadenylated nucleotide sequence to the 3′ end of saidRNA sequence; wherein the promoter sequence is heterologous with respectto the structural DNA sequence and causes sufficient expression of saidenzyme in a plant cell to enhance the phosphonate herbicide tolerance ofa plant cell transformed with said polynucleotide sequence, and whereinsaid plant cell expresses a GOX gene which encodes a glyphosateoxidoreductase enzyme; b) selecting a transformed plant cell; and c)regenerating from the transformed plant cell a genetically transformedplant which exhibits improved phosphonate herbicide tolerance.
 25. Themethod according to claim 24, wherein a transformed plant cell isselected by an ability to grow in the presence of a selective agent,wherein said agent is selected from the group consisting of glyphosateand AMPA.
 26. A peptide comprising an acyltransferase enzyme thatcatalyzes the transfer of an acyl group from an acylated donor to theterminal amine of a phosphonate herbicide.
 27. The peptide according toclaim 26 wherein the acyltransferase enzyme is comprised of a sequenceselected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 8, SEQ IDNO: 12, and SEQ ID NO:
 20. 28. The peptide according to claim 26 whereinsaid phosphonate herbicide is selected from the group consisting ofglyphosate and AMPA.
 29. The peptide according to claim 26 wherein theacyltransferase enzyme is expressed in a cell from a DNA sequence whichis or hybridizes to a polynucleotide sequence selected from the groupconsisting of SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11, and SEQ ID NO:19.
 30. The peptide according to claim 27 wherein the DNA sequence isderived from a microbe, said microbe being a member of the genusselected from the group consisting of Enterobacteriaceae, Streptomyces,Bacillus, Actinobacillus, Ascomycota, and Basidiomycota.
 31. A methodfor selecting one or more cells transformed with a vector containing anacyltransferase gene encoding an enzyme which functions to N-acetylate aphosphonate herbicide compound comprising the steps of: a) transforminga population of cells with said vector; b) incubating said transformedcells in the presence of an inhibitory amount of phosphonate herbicidecompound; c) identifying one or more cells that grow in the presence ofsaid inhibitory amount of said compound; and d) isolating and purifyingsaid one or more cells that grow in the presence of said inhibitoryamount of said compound.
 32. A vector according to claim 31 comprisingan acyltransferase gene encoding an enzyme which functions in said oneor more cells to transfer an acyl group from an acylated donor substrateto the terminal amine of a phosphonate herbicide compound.
 33. Thevector according to claim 31 wherein the gene is expressed in a hostcell.
 34. The vector according to claim 33 wherein the host cell is notinhibited by the presence of a phosphonate herbicide in an amount whichis inhibitory to a host cell lacking a functional acyltransferase geneencoding said enzyme.
 35. The host cell according to claim 34 selectedfrom the group consisting of bacterial cells, fungal cells, animalcells, and plant cells.
 36. The host cell according to claim 35 whereinthe bacterial cell is selected from the bacterial species consisting ofEnterobacteriaceae, Mycobacteriaceae, Agrobacteriaceae,Actinobacteriaceae, Streptomyces, and Bacillus.
 37. The host cellaccording to claim 35 wherein the fungal cell is selected from thefungal species consisting of Ascomycota, Basidiomycota, andDeuteromycota.
 38. The host cell according to claim 35 wherein the plantcell is selected from the plant species consisting of Glycine max, Zeamays, Nicotania tabacum, Gossypium gossypia, Triticum aestivum, andBrassica napus.
 39. An antibody which binds to an acyltransferaseprotein sequence, wherein said acyltransferase is selected from thegroup consisting of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, and SEQID NO:
 20. 40. A method for identifying a recombinant acyltransferasegene in a sample comprising a) providing one or more distinctpolynucleotide sequences capable of hybridizing to said gene; b)providing a reference sample comprising one or more polynucleotidesequences complementary to said distinct polynucleotide sequences; andc) providing instructions for combining said distinct sequences, saidreference sample, and said recombinant acyltransferase gene in a sample,and d) detecting the recombinant gene in said sample.
 41. A kit fordetecting the presence of a recombinant acyltransferase gene in a samplecomprising a) providing one or more distinct polynucleotide sequencescapable of hybridizing to said gene; b) providing a reference samplecomprising one or more polynucleotide sequences complementary to saiddistinct polynucleotide sequences; and c) providing instructions forcombining said distinct sequences, said reference sample, and saidrecombinant acyltransferase gene in a sample packaged together in a kit.42. A plant comprising a polynucleotide sequence containing a gene whichencodes an acetyltransferase protein, wherein expression of said gene insaid plant stimulates said plant's growth.